Loadable polymeric particles for enhanced imaging in clinical applications and methods of preparing and using the same

ABSTRACT

Particles are provided for use in therapeutic and/or diagnostic procedures. The particles include poly[bis(trifluoroethoxy)phosphazene] and/or a derivatives thereof which may be present throughout the particles or within an outer coating of the particles. The particles can also include a core having a hydrogel formed from an acrylic-based polymer. Such particles may be provided to a user in specific selected sizes to allow for selective embolization of certain sized blood vessels or localized treatment with an active component agent in specific clinical uses. Microspheres of the present invention may further be provided with physical and/or chemical enhancements within the particles&#39; cores to enhance visualization of the embolized tissue using a variety of medical imaging modalities, including conventional radiography, fluoroscopy, tomography, computerized tomography, ultrasound, scintillation, magnetic resonance, or other imaging technologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/257,535, filed Oct. 25, 2005, which claims the benefit ofpriority under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationsNo. 60/684,307, filed May 24, 2005 and 60/621,729, filed Oct. 25, 2004,the entire disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Small particles, including microspheres and nanospheres, have manymedical uses in diagnostic and/or therapeutic procedures. In selectedclinical applications, it may be advantageous to provide suchmicrospheres and nanospheres with physical and/or chemical qualities toenhance their visual identification using various clinical imagingmodalities to make the in vivo location of such microspheres apparent toa user. Such microspheres and nanospheres may allow for selectiveembolization of certain sized blood vessels under visual control usingvarious clinical imaging modalities in specific clinical uses.

Most particles used in medical applications are characterized bynumerous disadvantages including irritation of the tissues with whichthey come in contact and initiation of adverse immune reactions.Additionally, many of the materials used to prepare these particles maydegrade relatively rapidly within the mammalian body, thereby detractingfrom their utility in certain procedures where long term presence ofintact particles may be necessary. Moreover, the degradation of thesematerials may release toxic or irritating compounds causing adversereactions in the patients.

Some known particle types suffer from difficulties in achievingdesirable suspension properties when such particles are incorporatedinto a delivery suspension for injection into a site in the body to betreated. Many times, the particles settle out or tend to “float” in thesolution such that they are not uniformly suspended for even delivery.Furthermore, particles may tend to aggregate within the deliverysolution and/or adhere to some part of the delivery device, making itnecessary to compensate for these adhesive/attractive forces.

In order to achieve a stable dispersion, suitable dispersing agents maybe added, which may include surfactants directed at breaking downattractive particle interactions. Depending on the nature of theparticle interaction, materials such as the following may be used:cationic, anionic, or nonionic surfactants such as Tween™ 20, Tween™ 40,Tween™ 80, polyethylene glycols, sodium dodecyl sulfate, variousnaturally occurring proteins such as serum albumin, or any othermacromolecular surfactants in the delivery formulation. Furthermorethickening agents can be used help prevent particles from settling bysedimentation and to increase solution viscosity, for example, polyvinylalcohols, polyvinyl pyrrolidones, sugars, or dextrins. Density additivesmay also be used to achieve buoyancy.

It can also be difficult to visualize microparticles in solution todetermine their degree of suspension when using clear, transparentpolymeric acrylate hydrogel beads in aqueous suspension. The inertprecipitate barium sulfate may be used in particle form an additive forbone cement, for silicones, for rendering items visible during X-rayexamination and for providing radiopacity to polymeric acrylateparticles. See Jayakrishnan et al., Bull. Mat. Sci., Vol. 12, No. 1, pp.17-25 (1989). Barium sulfate also is known for improving fluidization,and is often used as an inorganic filler to impart anti-stick behaviorto moist, aggregated particles. Other attempts to increase visualizationof microparticles include the use of gold, for example, in EmbosphereGoIdTM, which provides a magenta color to acrylate microparticles usingsmall amounts of gold.

In certain medical applications, it may be of further value to providemicroparticles such as microspheres in one or more sizes. Furthermore,it may also be of value to provide each of such sizes of microspheresincorporated with color-coded associated dyes to indicate themicrosphere size to the user. In yet other applications of use, it mayfurther be of value to provide sized and color-coded microspheres to auser in similarly color-coded syringes or other containers for transportand delivery to further aid a user in identifying the size ofmicrospheres being used.

There thus exists in the art a need for small particles that can beformed to have a preferential generally spherical configuration forcertain applications such as various therapeutic and diagnosticprocedures which are not degraded by the natural systems of themammalian system, are biocompatible, are easy to visualize in suspensionwhile in use and/or demonstrate acceptable physical and suspensionproperties.

BRIEF SUMMARY OF THE INVENTION

This invention includes a particle for use in a therapeutic and/ordiagnostic clinical procedure. The particle comprisespoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof.

The present invention further includes particles comprisingpoly[bis(trifluoroethoxy)phosphazene and/or a derivative thereofprovided as microspheres provided in one or more specified sizes.

The present invention further includes particles comprisingpoly[bis(trifluoroethoxy)phosphazene and/or a derivative thereofprovided as sized microspheres and further comprising a color-coded dyeincorporated into or attached to the exterior of the microspheres tovisually aid a user in identifying the size of microspheres in use.

Microspheres of the present invention may further be provided as sizedmicrospheres further comprising a color-coded dye incorporated into orattached to the exterior of the microspheres and contained or deliveredin a similarly color-coded syringe or other transport or deliverycontainer to further visually aid a user in providing a visualconfirmation of the specific size of microspheres in use.

Also included is a method of minimizing blood flow to a tissue in amammal comprising occluding at least a portion of a blood vessel of themammal with at least one particle, wherein the particle comprises apoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof.

Further described herein is a method of delivering an active agent to alocalized area within a body of a mammal comprising contacting thelocalized area with at least one of a particle comprisingpoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof and anactive agent, such that an effective amount of the active agent isexposed to the localized area.

Also within the invention is a sustained release formulation of anactive agent for oral administration, the formulation comprising apolymer capsule and an active agent, wherein the polymeric capsulecomprises poly[bis(trifluoroethoxy)phosphazene] and/or a derivativethereof.

The invention further includes a method of tracing the passage of aparticle through a blood vessel in a mammal, the method comprisinginjecting into the bloodstream of a mammal at least one tracer particle,the tracer particle comprising poly[bis(trifluoroethoxy)phosphazene]and/or a derivative thereof and a contrast agent, and imaging the routeof the particle.

Also further described herein is a method of delivering a contrast agentfor magnetic resonance imaging (MRI) to a localized area within a bodyof a mammal comprising contacting the localized area with at least oneof a particle comprising poly[bis(trifluoroethoxy)phosphazene] and/or aderivative thereof and a contrast agent for magnetic resonance imaging,such that the presence of such particles may be identified and evaluatedusing magnetic resonance imaging techniques.

The invention further includes a method of tracing the passage of aparticle through a blood vessel in a mammal, the method comprisinginjecting into the bloodstream of a mammal at least one tracer particle,the tracer particle comprising poly[bis(trifluoroethoxy)phosphazene]and/or a derivative thereof and a radioisotope, and imaging the route ofthe particle using scintillation or other radiation sensing imagingmodalities.

Additionally, a method of enhanced ultrasound imaging is describedherein. The method comprises administering to an ultrasound subject atleast one particle comprising poly[bis(trifluoroethoxy)phosphazene]and/or a derivative thereof and a core containing one or more gas filledmicrobubbles to an area of the ultrasound subject to enhanceechogenicity therein, and imaging the area of the subject usingultrasound.

The invention also includes a method of delivering an active agent to alocalized area within the body of a mammal comprising contacting thelocalized area with at least one of a particle comprisingpoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof and anactive agent, such that an effective amount of the active agent isexposed to the localized area, wherein the particle comprises an agentto increase density.

The invention also includes a method of delivering a radiographiccontrast agent to a localized area within the body of a mammalcomprising contacting the localized area with at least one of a particlecomprising poly[bis(trifluoroethoxy)phosphazene] and/or a derivativethereof and a radiographic contrast agent, such that the location of aplurality of the particles may be seen using conventional radiographicimaging techniques, including but not limited to plain radiographs,x-ray tomography, and computerized axial tomography.

Further, a method of minimizing agglomeration of particles formed fromacrylic-based polymers is described in which the method comprisesproviding barium sulfate to the core and/or surface of the particles.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments that are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows a schematic representation of a general cryoextractionscheme used to prepare particles according to one embodiment of theinvention;

FIG. 2 shows the manual dripping technique by which the polymer solutionwas supplied to liquid nitrogen in preparation of the microspheres ofExample 1, herein;

FIG. 3A and FIG. 3B show unloaded polyphosphazene particles(microspheres) as prepared by one embodiment of the cryoextractionmethod as described herein. FIG. 3A shows a 4× optical microscope viewand FIG. 3B shows a 100× scanning electron microscope view;

FIG. 4A and FIG. 4B show particles (microsphere) formed according to oneembodiment of the invention loaded with bovine insulin (20% (wt/wt)) at100× magnification SEM;

FIG. 5A and FIG. 5B show the surface morphology of unloadedpolyphosphazene microspheres. FIG. 5A is an image obtained using anatomic force microscope and FIG. 5B is a scanning electron micrographshowing the surface of an unloaded polyphosphazene microsphere at 5000×magnification;

FIGS. 6 and 7 show a cryoextraction setup for use in an embodiment ofthe invention wherein FIG. 6 is a cryoextraction vessel and FIG. 7 is asyringe pump;

FIG. 8 is a cross-sectional view of an apparatus for use inmicrocatheter testing of microparticles in Example 14 herein;

FIGS. 9A and 9B show an SEM at 1.0KX magnification of the surface of theSample C microparticles just after the hydration/dehydration cycle andat a 50.00KX magnification of the film thickness of microparticlesformed in accordance with Sample C of Example 12 used in the evaluationof Example 14, respectively;

FIGS. 10A, 10B, 10C and 10D are SEMs of microparticles made inaccordance with Sample C of Example 12 used in the evaluation of Example14 after passing through a catheter showing surface features (FIGS. 10A,10B and 10C) at 1.0KX magnification and at 5.0KX magnification (FIG.10D);

FIGS. 11A, 11B, 11C and 11D are SEMs of microparticles formed inaccordance with Sample C of Example 12 after thermal stress testing inExample 14. FIG. 11A is a 50× magnification of a minor amount ofdelamination in the strong white contrast portion. FIG. 11B is a 200×magnification of the microparticles of FIG. 11A. FIGS. 11C and 11D are,respectively, 200× and 1.0KX magnified SEMs of other Sample Cmicroparticles showing only minor defects.

FIGS. 12A and 12B illustrate various aspects the microparticles formedand used according to Examples 22 and 23. FIG. 12A is a conceptualrepresentation of a selective embolization of an exemplary tumor mass byintravascular administration of microspheres. FIG. 12B shows anexemplary particle of the present invention comprising apolyphosphazene-coated microsphere further comprising a core with agas-filled microbubble for enhanced visibility of sonography.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are particles that may be manufactured usingpoly[bis(trifluoroethoxy)phosphazene] and/or derivatives thereof, aswell as methods of preparing such particles. Additionally, describedherein are therapeutic and/or diagnostic methods and procedures whichuse the particles as described herein, including methods of embolizationusing the particles, methods of delivery of an active agent using theparticle (either orally or locally), methods of tracing or visualizingblood or other biological fluids through the body using the particles,methods of enhanced ultrasound (sonography) using the particles, and thelike.

Also included are sustained release drug delivery formulations for oraladministration including the particles for localized delivery of anactive agent to the gastrointestinal system and/or systemic delivery ofan active agent as well as a sustained release drug delivery formulationthat can be injected subcutaneously or intravenously for localizeddelivery of an active agent.

All of the methods, compositions and formulations of the inventionutilize at least one particle as described herein. “Particle” and“particles” as used herein mean a substantially spherical or ellipsoidarticle(s), hollow or solid, that may have any diameter suitable for usein the specific methods and applications described below, including amicrosphere(s) and a nanosphere(s), beads and other bodies of a similarnature known in the art.

The preferred particles of the invention according to one embodimentdescribed herein are composed, in whole or in part, the specificpolyphosphazene polymer known as poly[bis(trifluoroethoxy)phosphazene]or a derivative of poly[bis(trifluoroethoxy)phosphazene].

Use of this specific polymer provides particles that are at least inpart inorganic in that they include an inorganic polymer backbone andwhich are also biocompatible in that when introduced into a mammal(including humans and animals), they do not significantly induce aresponse of the specific or non-specific immune systems. The scope ofthe invention also includes the use(s) of such particles as controlleddrug delivery vehicles or tracer particles for the visualization ofblood vessels and other organs.

The particles are useful in a variety of therapeutic and/or diagnosticprocedures in part because they can be prepared in sizes large enough toocclude a blood vessel as well as small enough to easily pass throughthe smaller vessels, for example, visualization or drug deliverypurposes. Additionally, owing to the biocompatible nature of thepolymer, the particles facilitate avoidance or elimination ofimmunogenic reactions generally encountered when foreign bodies areintroduced into a mammalian body, such as “implant rejection” or“allergic shock,” and other adverse reactions of the immune system.Moreover, it has been found that the particles of the invention exhibitreduced biodegradation in vivo, thereby increasing the long-termstability of the particle in the biological environment. Moreover, inthose situations where some degradation is undergone by the polymer inthe particle, the products released from the degradation include onlynon-toxic concentrations of phosphorous, ammonia, and trifluoroethanol,which, advantageously, is known to promote anti-inflammatory responseswhen in contact with mammalian tissue.

Each of the particles in the invention is formed at least in part of thepolymer, poly[bis(2,2,2-trifluoroethoxy)phosphazene] or a derivativethereof (referred to further herein as“poly[bis(trifluoroethoxy)phosphazene]”. As described herein, thepolymer poly[bis(2,2,2-trifluoroethoxy)phosphazene] or derivativesthereof have chemical and biological qualities that distinguish thispolymer from other know polymers in general, and from other knowpolyphosphazenes in particular. In one aspect of this invention, thepolyphosphazene is poly[bis(2,2,2-trifluoroethoxy)phosphazene] orderivatives thereof, such as other alkoxide, halogenated alkoxide, orfluorinated alkoxide substituted analogs thereof. The preferredpoly[bis(trifluoroethoxy)phosphazene]polymer is made up of repeatingmonomers represented by the formula (I) shown below:

wherein R¹ to R⁶ are all trifluoroethoxy (OCH₂CF₃) groups, and wherein nmay vary from at least about 40 to about 100,000, as disclosed herein.Alternatively, one may use derivatives of this polymer in the presentinvention. The term “derivatives” is meant to refer to polymers made upof monomers having the structure of formula I but where one or more ofthe R¹ to R⁶ functional group(s) is replaced by a different functionalgroup(s), such as an unsubstituted alkoxide, a halogenated alkoxide, afluorinated alkoxide, or any combination thereof, or where one or moreof the R¹ to R⁶ is replaced by any of the other functional group(s)disclosed herein, but where the biological inertness of the polymer isnot substantially altered.

In one aspect of the polyphosphazene of formula (I) illustrated above,for example, at least one of the substituents R¹ to R⁶ can be anunsubstituted alkoxy substituent, such as methoxy (OCH₃), ethoxy(OCH₂CH₃) or n-propoxy (OCH₂CH₂CH₃). In another aspect, for example, atleast one of the substituents R¹ to R⁶ is an alkoxy group substitutedwith at least one fluorine atom. Examples of useful fluorine-substitutedalkoxy groups R¹ to R⁶ include, but are not limited to OCF₃, OCH₂CF₃,OCH₂CH₂CF₃, OCH₂CF₂CF₃, OCH(CF₃)₂, OCCH₃(CF₃)₂, OCH₂CF₂CF₂CF₃,OCH₂(CF₂)₃CF₃, OCH₂(CF₂)₄CF₃, OCH₂(CF₂)₅CF₃, OCH₂(CF₂)₆CF₃,OCH₂(CF₂)₇CF₃, OCH₂CF₂CHF₂, OCH₂CF₂CF₂CHF₂, OCH₂(CF₂)₃CHF₂,OCH₂(CF₂)₄CHF₂, OCH₂(CF₂)₅CHF₂, OCH₂(CF₂)₆CHF₂, OCH₂(CF₂)₇CHF₂, and thelike. Thus, while trifluoroethoxy (OCH₂CF₃) groups are preferred, thesefurther exemplary functional groups also may be used alone, incombination with trifluoroethoxy, or in combination with each other. Inone aspect, examples of especially useful fluorinated alkoxidefunctional groups that may be used include, but are not limited to,2,2,3,3,3-pentafluoropropyloxy (OCH₂CF₂CF₃),2,2,2,2′,2′,2′-hexafluoroisopropyloxy (OCH(CF₃)₂),2,2,3,3,4,4,4-heptafluorobutyloxy (OCH₂CF₂CF₂CF₃),3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyloxy (OCH₂(CF₂)₇CF₃),2,2,3,3,-tetrafluoropropyloxy (OCH₂CF₂CHF₂),2,2,3,3,4,4-hexafluorobutyloxy (OCH₂CF₂CF₂CHF₂),3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorooctyloxy (OCH₂(CF₂)₇CHF₂), and thelike, including combinations thereof.

Further, in some embodiments, 1% or less of the R¹ to R⁶ groups may bealkenoxy groups, a feature that may assist in crosslinking to provide amore elastomeric phosphazene polymer. In this aspect, alkenoxy groupsinclude, but are not limited to, OCH₂CH═CH₂, OCH₂CH₂CH═CH₂, allylphenoxygroups, and the like, including combinations thereof. Also in formula(I) illustrated herein, the residues R¹ to R⁶ are each independentlyvariable and therefore can be the same or different.

By indicating that n can be as large as Go in formula I, it is intendedto specify values of n that encompass polyphosphazene polymers that canhave an average molecular weight of up to about 75 million Daltons. Forexample, in one aspect, n can vary from at least about 40 to about100,000. In another aspect, by indicating that n can be as large as ∞ informula I, it is intended to specify values of n from about 4,000 toabout 50,000, more preferably, n is about 7,000 to about 40,000 and mostpreferably n is about 13,000 to about 30,000.

In another aspect of this invention, the polymer used to prepare thepolymers disclosed herein has a molecular weight based on the aboveformula, which can be a molecular weight of at least about 70,000 g/mol,more preferably at least about 1,000,000 g/mol, and still morepreferably a molecular weight of at least about 3×10⁶ g/mol to about20×10⁶ g/mol. Most preferred are polymers having molecular weights of atleast about 10,000,000 g/mol.

In a further aspect of the polyphosphazene formula (I) illustratedherein, n is 2 to ∞, and R¹ to R⁶ are groups which are each selectedindependently from alkyl, aminoalkyl, haloalkyl, thioalkyl, thioaryl,alkoxy, haloalkoxy, aryloxy, haloaryloxy, alkylthiolate, arylthiolate,alkylsulphonyl, alkylamino, dialkylamino, heterocycloalkyl comprisingone or more heteroatoms selected from nitrogen, oxygen, sulfur,phosphorus, or a combination thereof, or heteroaryl comprising one ormore heteroatoms selected from nitrogen, oxygen, sulfur, phosphorus, ora combination thereof. In this aspect of fonnula (I), the pendant sidegroups or moieties (also termed “residues”) R¹ to R⁶ are eachindependently variable and therefore can be the same or different.Further, R¹ to R⁶ can be substituted or unsubstituted. The alkyl groupsor moieties within the alkoxy, alkylsulphonyl, dialkylamino, and otheralkyl-containing groups can be, for example, straight or branched chainalkyl groups having from 1 to 20 carbon atoms, typically from 1 to 12carbon atoms, it being possible for the alkyl groups to be furthersubstituted, for example, by at least one halogen atom, such as afluorine atom or other functional group such as those noted for the R¹to R⁶ groups above. By specifying alkyl groups such as propyl or butyl,it is intended to encompass any isomer of the particular alkyl group.

In one aspect, examples of alkoxy groups include, but are not limitedto, methoxy, ethoxy, propoxy, and butoxy groups, and the like, which canalso be further substituted. For example the alkoxy group can besubstituted by at least one fluorine atom, with 2,2,2-trifluoroethoxyconstituting a useful alkoxy group. In another aspect, one or more ofthe alkoxy groups contains at least one fluorine atom. Further, thealkoxy group can contain at least two fluorine atoms or the alkoxy groupcan contain three fluorine atoms. For example, the polyphosphazene thatis combined with the silicone can bepoly[bis(2,2,2-trifluoroethoxy)phosphazene]. Alkoxy groups of thepolymer can also be combinations of the aforementioned embodimentswherein one or more fluorine atoms are present on the polyphosphazene incombination with other groups or atoms.

Examples of alkylsulphonyl substituents include, but are not limited to,methylsulphonyl, ethylsulphonyl, propylsuiphonyl, and butylsulphonylgroups. Examples of dialkylamino substituents include, but are notlimited to, dimethyl-, diethyl-, dipropyl-, and dibutylamino groups.Again, by specifying alkyl groups such as propyl or butyl, it isintended to encompass any isomer of the particular alkyl group.

Exemplary aryloxy groups include, for example, compounds having one ormore aromatic ring systems having at least one oxygen atom,non-oxygenated atom, and/or rings having alkoxy substituents, it beingpossible for the aryl group to be substituted for example by at leastone alkyl or alkoxy substituent defined above. Examples of aryloxygroups include, but are not limited to, phenoxy and naphthoxy groups,and derivatives thereof including, for example, substituted phenoxy andnaphthoxy groups.

The heterocycloalkyl group can be, for example, a ring system whichcontains from 3 to 10 atoms, at least one ring atom being a nitrogen,oxygen, sulfur, phosphorus, or any combination of these heteroatoms. Thehetereocycloalkyl group can be substituted, for example, by at least onealkyl or alkoxy substituent as defined above. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, and morpholinyl groups, and substitutedanalogs thereof.

The heteroaryl group can be, for example, a compound having one or morearomatic ring systems, at least one ring atom being a nitrogen, anoxygen, a sulfur, a phosphorus, or any combination of these heteroatoms.The heteroaryl group can be substituted for example by at least onealkyl or alkoxy substituent defined above. Examples of heteroaryl groupsinclude, but are not limited to, imidazolyl, thiophene, furan, oxazolyl,pyrrolyl, pyridinyl, pyridinolyl, isoquinolinyl, and quinolinyl groups,and derivatives thereof, such as substituted groups.

The diameter of a particle formed according to the invention will varydepending on the end application in which the particle is to be used.The diameter of such particles is preferably about 1 to about 5,000 μm,with a diameter of about 1 to about 1,000 μm being most preferred. Otherpreferred sizes include diameters of about 200 to about 500 μm, about 1to about 200 μm and greater than about 500 μm. In methods using theparticle where more than one particle is preferred it is not necessarythat all particles are of the same diameter or shape.

The particles may also include other compounds which function toenhance, alter or otherwise modify the behavior of the polymer orparticle either during its preparation or in its therapeutic and/ordiagnostic use. For example, active agents such as peptides, proteins,hoiiuones, carbohydrates, polysaccharides, nucleic acids, lipids,vitamins, steroids and organic or inorganic drugs may be incorporatedinto the particle. Excipients such as dextran, other sugars,polyethylene glycol, glucose, and various salts, including, for example,chitosan glutamate, may be included in the particle.

Additionally, if desired, polymers other than thepoly[bis(trifluoroethoxy)phosphazene] and/or its derivative may beincluded with in the particle. Examples of polymers may includepoly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone),polycarbonates, polyamides, polyanhydrides, polyamino acids,polyorthoesters, polyacetals, polycyanoacrylates, and polyurethanes.Other polymers include polyacrylates, ethylene-vinyl acetateco-polymers, acyl substituted cellulose acetates and derivativesthereof, degradable or non-degradable polyurethanes, polystyrenes,polyvinylchloride, polyvinyl fluoride, poly(vinyl imidazole),chlorosulphonated polyolefins, and polyethylene oxide. Examples ofpolyacrylates include, but are not limited to, acrylic acid, butylacrylate, ethylhexyl acrylate, methyl acrylate, ethyl acrylate,acrylonitrile, methyl methacrylate, TMPTA (trimethylolpropanetriacrylate), and the like. One may incorporate the selected compoundsby any means known in the art, including diffusing, inserting orentrapping the additional compounds in the matrix of an already formedparticle or by adding the additional compound to a polymer melt or to apolymer solvent in the preparation of the particle such as describedherein.

The loaded or unloaded particle may be coated with an additional polymerlayer or layers, including polymers such as those mentioned hereinabove.Further, poly[bis(trifluoroethoxy)phosphazene or its derivatives may beused to form such a coating on a particle formed of other suitablepolymers or copolymers known or to be developed in the art that are usedto form particles as described herein. Preferably, when coating aparticle such as a microparticle, poly[bis(trifluoroethoxy)phosphazeneis applied as a coating on a microparticle(s) formed of an acrylic-basedpolymer as set forth in further detail below.

Coatings are beneficial, for example, if the particle(s) are to be usedin a sustained release, orally administered, drug delivery formulation(enteric coating) or if the particles are to be loaded with apotentially toxic contrast agent (non-biodegradable coating).

The microspheres may be prepared by any means known in the art that issuitable for the preparation of particles containingpoly[bis(trifluoroethoxy)phosphazene]. In a procedure according to anembodiment herein a “polymer solution” is prepared by mixing one or morepolymer solvent(s) and the poly[bis(trifluoroethoxy)phosphazene and/or aderivative thereof until the polymer is dissolved.

Suitable solvents for use in the preparation of the polymer solutioninclude any in which the polymer poly[bis(trifluoroethoxy)phosphazeneand/or its derivatives are soluble. Exemplary solvents include, withoutlimitation, ethyl-, propyl-, pentyl-, octylacetate, acetone,methylethylketone, methylpropylketone, methylisobutylketone,tetrahydrofurane, cyclohexanone, dimethylacetamide, acetonitrile,dimethyl ether, hexafluorobenzene or combinations thereof.

The polymer solution contains the poly[bis(trifluoroethoxy)phosphazeneand/or its derivative polymer in a concentration of about 1% by weightof polymer to 20% by weight of polymer, preferably about 5% to 10% byweight of polymer. Other polymers, as discussed above, may be present inthe solution, or may be added to the vessel in the form of a secondsolution powder or other form, if one wishes to include such polymers inthe final particle.

In carrying out the process, the polymer solution is next dispensed,preferably in the form of drops or an aerosol, into a vessel containinga non-solvent. By “non-solvent” it is meant any organic or inorganicsolvents that do not substantially dissolve thepoly[bis(trifluoroethoxy)phosphazene polymer and which have a meltingpoint that is lower relative to the melting point of the solvent inwhich the polymer is dissolved (“polymer solvent”), so that thenon-solvent thaws before the solvent thaws in the course of theincubation step. Preferably, this difference between the melting pointof the non-solvent and the polymer solvent is about 10° C., morepreferably about 15° C., and most preferably, greater than about 20° C.Under certain conditions it has been found that the structural integrityof the resultant particle may be enhanced if the difference of themelting points of the polymer solvent and of the non-solvent is greaterthan 15° C. However, it is sufficient that the non-solvent point ismerely slightly lower than that of the polymer solvent.

The non-solvent/polymer solvent combination is incubated forapproximately 1 to 5 days or until the polymer solvent has beencompletely removed from the particles. While not wishing to be bound bytheory, it is hypothesized that during the incubation, the non-solventfunctions to extract the polymer solvent from the microscopic polymersolution droplets from the particles such that the polymer is at leastgelled. As the incubation period passes, the droplets will shrink andthe solvent becomes further extracted, leading to a hardened outerpolymeric shell containing a gelled polymer core, and finally, aftercompletion of the incubation, a complete removal of the residualsolvent. To ensure that the polymeric droplets retain a substantiallyspherical shape during the incubation period, they are maintained in afrozen or substantially gelled state during most if not all of theincubation period. Therefore, the non-solvent temperature may stay belowthe melting point of the solvent during the cryoextraction process.

As shown in FIG. 1, at the vessel labeled (a), polymer solution dropletsare shown being dispensed either with a syringe or other device at acontrolled rate onto a top layer of liquid nitrogen. The nitrogen layeris situated over a bottom layer consisting of the selected non-solvent,which will eventually serve to extract the solvent from the frozenpolymer solution droplets. The non-solvent layer has been previouslyfrozen with liquid nitrogen prior to the dispensing of the polymersolution. The vessel labeled (b) shows the onset of the dewing of thefrozen nonsolvent, into which the frozen polymeric droplets will sink.The vessel labeled (c) shows the cryoextraction procedure afterapproximately three days of incubation wherein the polymer solutiondroplets, incubated within the non-solvent, have been depleted of asubstantial amount of solvent. The result is a gelled, polymericparticle in the form of a bead having a hardened outer shell. As can beseen by the representation, the non-solvent height within the vessel isslightly reduced due to some evaporation of the non-solvent. The size ofthe beads will shrink quite substantially during this process dependingon the initial concentration of the polymer in the polymer solution.

In one embodiment of a method of preparing apoly[bis(trifluoroethoxy)phosphazene-containing particle(s) according tothe invention, such particles can be formed using any method known or tobe developed in the art. Two exemplary preferred methods ofaccomplishing this include wherein (i) the non-solvent residing in thevessel in the method embodiment described above is cooled to close toits freezing point or to its freezing point prior to the addition of thepolymer solution such that the polymer droplets freeze upon contact withthe pre-cooled non-solvent; or (ii) the polymer droplets are frozen bycontacting them with a liquefied gas such as nitrogen, which is placedover a bed of pre-frozen non-solvent (see, FIG. 2). In method (ii),after the nitrogen evaporates, the non-solvent slowly thaws and themicrospheres in their frozen state will sink into the liquid, coldnon-solvent where the extraction process (removal of the polymersolvent) will be carried out.

By modifying this general process, one may prepare particles that arehollow or substantially hollow or porous. For example, if the removal ofthe solvent from the bead is carried out quickly, for example, byapplying a vacuum during the final stage of incubation, porous beadswill result.

The particles of the invention can be prepared in any size desired,“Microspheres” may be obtained by nebulizing the polymer solution into apolymer aerosol using either pneumatic or ultrasonic nozzles, such as,for example a Sonotek 8700-60 ms or a Lechler US50 ultrasonic nozzle,each available from Sono[.tek] Corporation, Milton, N.Y., U.S.A. andLechler GmbH, Metzingen, Germany. Larger particles may be obtained bydispensing the droplets into the non-solvent solution using a syringe orother drop-forming device. Moreover, as will be known to a person ofskill in the art, the size of the particle may also be altered ormodified by an increase or decrease of the initial concentration of thepolymer in the polymer solution, as a higher concentration will lead toan increased sphere diameter.

In an alternative embodiment of the particles described herein, theparticles can include a standard and/or a preferred core based on anacrylic polymer or copolymer with a shell ofpoly[bis(trifluoroethoxy)phosphazene. Such particles can provide apreferred spherical shape and improved specific gravity for use in asuspension of contrast media for embolization. The acrylic polymer basedpolymers with poly[bis(trifluoroethoxy)phosphazene shell describedherein provide a substantially spherical shape, mechanical flexibilityand compressibility, improved specific gravity properties. The corepolymers may be formed using any acceptable technique known in the art,such as that described in B. Thanoo et al., “Preparation of HydrogelBeads from Crosslinked Poly(Methyl Methacrylate) Microspheres byAlkaline Hydrolysis,” J. Appl. P. Sci., Vol. 38, 1153-1161 (1990),incorporated herein by reference with respect thereto. Suchacrylic-based polymers are preferably formed by polymerizingunhydrolyzed precursors, including, without limitation, methyl acrylate(MA), methyl methacrylate (MMA), ethylmethacrylate (EMA), hexamethyl(HMMA) or hydroxyethyl methacrylate (HEMA), and derivatives, variants orcopolymers of such acrylic acid derivatives. Most preferred is MMA. Thepolymer should be present in the core in a hydrated or partiallyhydrated (hydrogel) form. Such polymers are preferably cross-linked inorder to provide suitable hydrogel properties and structure, such asenhanced non-biodegradability, and to help retain the mechanicalstability of the polymer structure by resisting dissolution by water.

Preferably, the core prepolymers are formed by dispersion polymerizationthat may be of the suspension or emulsion polymerization type. Emulsionpolymerization results in substantially spherical particles of about 10nm to about 10 microns. Suspension polymerization results in similarparticles but of larger sizes of about 50 to about 1200 microns.

Suspension polymerization may be initiated with a thermal initiator,which may be solubilized in the aqueous or, more preferably, monomerphase. Suitable initiators for use in the monomer phase compositioninclude benzoyl peroxide, lauroyl peroxide or other similarperoxide-based initiators known or to be developed in the art, with themost preferred initiator being lauroyl peroxide. The initiator ispreferably present in an amount of about 0.1 to about 5 percent byweight based on the weight of the monomer, more preferably about 0.3 toabout 1 percent by weight based on the weight of the monomer. As notedabove, a cross-linking co-monomer is preferred for use in forming thehydrated polymer. Suitable cross-linking co-monomers for use with theacrylic-based principle monomer(s) used in preparing a polymerizedparticle core, include various glycol-based materials such as ethyleneglycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA)or most preferably, triethylene glycol dimethacrylate (TEGMDA). A chaintransfer agent may also be provided if desired. Any suitable MApolymerization chain transfer agent may be used. In the preferredembodiment herein, dodecylmercaptane may be used as a chain transferagent in amounts acceptable for the particular polymerization reaction.

The aqueous phase composition preferably includes asurfactant/dispersant as well as a complexing agent, and an optionalbuffer. Surfactants/dispersants should be compatible with the monomersused herein, including Cyanamer® 370M, polyacrylic acid and partiallyhydrolyzed polyvinyl alcohol surfactants such as 4/88, 26/88, 40/88. Adispersant should be present in an amount of about 0.1 to about 5percent by weight based on the amount of water in the dispersion, morepreferably about 0.2 to about 1 percent by weight based on the amount ofwater in the dispersion. An optional buffer solution may be used ifneeded to maintain adequate pH. A preferred buffer solution includessodium phosphates (Na₂HPO₄/NaH₂PO₄). A suitable complexing agent isethylene diamine tetraacetic acid (EDTA), which may be added to theaqueous phase in a concentration of from about 10 to about 40 ppm EDTA,and more preferably about 20 to about 30 ppm. It is preferred that inthe aqueous phase composition, the monomer to water ratio is about 1:4to about 1:6.

The polymerization should take place at about ambient conditions,preferably from about 60° C. to about 80° C. with a time to gelation ofabout one to two hours. Stirring at rates of 100 to 500 rpm is preferredfor particle formation, with lower rates applying to larger sizedparticles and higher rates applying to smaller sized particles.

Once PMMA (poly-MMA) particles, such as microparticles, are formed, theyare preferably subjected to hydrolysis conditions typical of those inthe art, including use of about 1-10 molar excess of potassium hydroxideper mol of PMMA. Such potassium hydroxide is provided in a concentrationof about 1-15% potassium hydroxide in ethylene glycol. The solution isthen heated preferably at temperatures of about 150-185° C. for severalhours. Alternatively, to minimize reactant amounts and cost, it ispreferred that lesser amounts of potassium hydroxide be used which areless than about 5 molar excess of potassium hydroxide per mole of PMMA,more preferably about 3 molar excess or less. For such hydrolyticreactions, a concentration of about 10-15% potassium hydroxide inethylene glycol is also preferably used, and more preferably about 14%to about 15%. It will be understood by one skilled in the art, thatheating conditions at higher temperatures may be used to decreaseoverall reaction times. Reaction times may be varied depending on theoverall diameter of the resultant particles. For example, the followingconditions are able to provide particles having about 35%compressibility and desired stability: for diameters of about 200-300μm, the solution should be heated for about 7.5 to about 8.5 hours; fordiameters of about 300-355 μm, about 9.5 to about 10.5 hours; fordiameters of about 355-400 μm, about 11.5 to about 12.5 hours; and forabout 400-455 μm, about 13.5 to about 14.5 hours, and the like. Theparticle size can be adjusted using variations in the polymerizationprocess, for example, by varying the stirring speed and the ratio of themonomer to the aqueous phase. Further, smaller sizes can be achieved byincreasing surfactant/dispersant ratio.

Following hydrolysis, particles are separated from the reaction mixtureand their pH may be adjusted to any range as suited for furtherprocessing steps or intended uses. The pH of the particle core may beadjusted in from about 1.0 to about 9.4, preferably about 7.4 ifintended for a physiological application. Since size, swelling ratio andelasticity of the hydrogel core material are dependent on pH value, thelower pH values may be used to have beneficial effects during drying toprevent particle agglomeration and/or structural damage. Particles arepreferably sieved into different size fractions according to intendeduse. Drying of particles preferably occurs using any standard dryingprocess, including use of an oven at a temperature of about 40°-80° C.for several hours up to about a day.

To provide desired surface properties to the hydrophilic hydrogelparticles, in order to provide adhesion for receiving apoly[bis(trifluoroethoxy)phosphazene coating, the surface of thehydrogel may be subjected to treatment with any suitable ionic ornon-ionic surfactant, such as tetraalkylammonium salts, polyalcohols andsimilar materials. A more peimanent change in adhesion properties isbrought about by rendering the surface of the particles hydrophobic byreaction of its polymethacrylic acid groups with a suitable reactant.Suitable reactants include, but are not limited to, hydrophobicalcohols, amides and carboxylic acid derivatives, more preferably theyinclude halogenated alcohols such as trifluoroethanol. Such surfacetreatment also prevents delamination of the coating from the core oncethe coating is applied. Preferred surface treatments may include,without limitation, an initial treatment with thionyl chloride followedby reaction with trifluoroethanol. Alternatively, the surface may betreated by suspending the particles in a mixture of sulfuric acid and ahydrophobic alcohol, such as trifluoroethanol. Such treatments arepreferred if the particles are to be coated in that they minimize anydelamination of a coating.

Alternatively, in some preferred embodiments of the present invention,the PMA (poly-MA) core particles may be coated with a surface layer ofand/or infused with barium sulfate. The barium sulfate is radio-opaqueand aids in visualization of the finished particles when in use. It alsoprovides enhanced fluidization properties to the particles such that itreduces agglomeration especially during drying and allows for fluid bedcoating of the PMA particles with an outer coating ofpoly[bis(trifluoroethoxy)phosphazene, thereby providing improvedadhesion between a poly[bis(trifluoroethoxy)phosphazene outer core and apolymeric acrylate core particles. By allowing fluidization even whenthe core particles are swollen, barium sulfate also improves the overallcoating and adhesion properties. By enabling the coating of the coreparticles even in a swollen state withpoly[bis(trifluoroethoxy)phosphazene, barium sulfate also reduces thepotential tendency of the poly[bis(trifluoroethoxy)phosphazene shells tocrack or rupture in comparison with coating the particles in a dry stateand then later exposing the particles to a suspension in which the coreparticles swell and exert force on the shell ofpoly[bis(trifluoroethoxy)phosphazene. A coating of barium sulfate on thecore particles is preferably applied by adhesion of the barium sulfatein the form of an opaque coating on the hydrogel surface of the PMAbeads. Barium sulfate can further assist in reducing electrostaticeffects that limit particle size. By allowing for absorption ofadditional humidity, the barium sulfate tends to counteract theelectrostatic effects.

Barium sulfate crystals adhering only loosely to the PMA particles maybe covalently crosslinked or chemically grafted to the particle surfaceby spraycoating a sufficient amount of an aminosilane adhesion promoteronto the PMA particle. This will help to effectively reduce bariumsulfate particulate matter in solution after hydration of the particles.Exemplary particles include 3-aminopropyl-trimethoxysilane and similarsilane-based adhesion promoters, such as, for example,N-methyl-aza-2,2,4-trimethylsilacyclopentane,2,2-dimethoxy-1,6-diaza-2-silacyclooctane,(3-trimethoxysilylpropyl)diethylene triamine,N-(3-(trimethoxysilyl)propyl)methanediamine,N¹,N²-bis(3-(trimethoxysilyl)propyl)ethane-1,2-diamine,1,3,5-tris(3-(trimethoxysilyl)propyl)-1,3,5-triazinane-2-4-6-trione, andsimilar silane-based adhesion promoters.

In various embodiments of the present invention, polyphosphazene-coatedmicrospheres may be provided with a hydrogel core containing variouscontrast agents suitable for enhanced visualization of such microspheresin vivo using magnetic resonance imaging (MRI), or derivativetechnologies thereof. Such contrast agents may include, but are notlimited to, agents that include tantalum, gadolinium, samarium, andother agents that are known to the art. Examples of MRI contrast agentsinclude, but are not limited to, ferric chloride, ferric ammoniumcitrate, gadolinium-DTPA (Gd-DTPA) with and without mannitol, Gd-DOTA,Gd-EDTA, GdCl₃, Gadodiamide, Gadoteridol, gadopentetate dimeglumine,Cr(III) agents, Mn(III)TPPS4 (manganese(III)tetra[4-sulfanatophenyl]porphyrin), Fe(III)TPPS4, manganese dichloride,Fe-EHPG (iron(III) ethylenebis-(2-hydroxyphenylglycine)),^(99m)Tc-iminodiacetate (Tc-IDA), chromium diethyl HIDA meglumine(Cr-HIDA), Gd-BOPTA (gadobenate dimeglumine), manganese(II)-dipyridoxaldiphosphate (Mn-DPDP), gadolinium oxide, superparamagnetic iron oxides(SPIO, also “small particle iron oxides”), ultrasmall supermagneticparticle iron oxides (USPIO, also “ultrasmall particle iron oxides”),and the like. One aspect of the present invention affords a method ofreducing the toxicity of various agents such as contrast agents likeFe(III)TPPS4, by containing the agent within the hydrogel core of themicrospheres disclosed herein.

In various embodiments of the present invention, polyphosphazene-coatedmicrospheres may be provided with a hydrogel core containing rare earthcompounds with paramagnetic qualities suitable for enhancedvisualization of such microspheres in vivo using magnetic resonanceimaging, paramagnetic transition methods, or derivative technologiesthereof known in the art.

Constituent atoms or molecules of paramagnetic materials have permanentmagnetic moments (dipoles), even in the absence of an applied field.This generally occurs due to the presence of unpaired electrons in theatomic/molecular electron orbitals. In pure paramagnetism, the dipolesdo not interact with one another and are randomly oriented in theabsence of an external field due to thermal agitation, resulting in zeronet magnetic moment. When a magnetic field is applied, the dipoles willtend to align with the applied field, resulting in a net magnetic momentin the direction of the applied field. In the classical description,this alignment can be understood to occur due to a torque being providedon the magnetic moments by an applied field, which tries to align thedipoles parallel to the applied field. However, the truer origins of thealignment can only be understood via the quantum-mechanical propertiesof spin and angular momentum.

If there is sufficient energy exchange between neighboring dipoles theywill interact, and may spontaneously align or anti-align and formmagnetic domains, resulting in ferromagnetism (permanent magnets) orantiferromagnetism, respectively. In general, paramagnetic effects arequite small: the magnetic susceptibility is of the order of 10⁻³ to 10⁻⁵for most paramagnets, but may be as high as 10⁻¹ for syntheticparamagnets such as ferrofluids.

For low levels of magnetization, the magnetization of paramagnetic rareearth compounds is approximated by Curie's law:

M=C(B/T)

where:

-   M is the resulting magnetization,-   B is the magnetic flux density of the applied field, measured in    tesla,-   T is absolute temperature, measured in Kelvin,-   C is a material-specific Curie constant.

Curie's law indicates that the susceptibility of paramagnetic materialsis inversely proportional to their temperature. However, Curie's law isonly valid under conditions of low magnetization, since it does notconsider the saturation of magnetization that occurs when the atomicdipoles are all aligned in parallel (after everything is aligned,increasing the external field will not increase the total magnetizationsince there can be no further alignment).

A further alternative for improving visualization of microparticles madeas noted herein include the absorption of a water soluble organic dyeinside the hydrogel core particles. Exemplary dyes are preferably thoseFDA dyes approved for human use and which are known or to be developedfor safe, non-toxic use in the body and which are capable of providingacceptable contrast. Organic dyes may include dyes such as D&C Violetno. 2 and others preferably approved for medical device uses, such asfor contact lenses and resorbable sutures. Whereas barium sulfateoperates as an inorganic filler and finely dispersed pigment that makesthe particles visible by light diffraction due to small crystal size,the dyes when impregnated in the particles absorb the complementary partof the visible color spectrum.

Particles, including microparticles made in accordance with theforegoing process for forming a core hydrogel polymer are then coatedwith poly[bis(trifluoroethoxy)phosphazene and/or its derivatives. Anysuitable coating process may be used, including solvent fluidized bedand/or spraying techniques. However, preferred results may be achievedusing fluidized bed techniques in which the particles pass through anair stream and are coated through spraying while they spin within theair stream. The poly[bis(trifluoroethoxy)phosphazene or derivativepolymer is provided in dilute solution for spraying to avoid clogging ofthe nozzle.

Exemplary solvents for use in such solutions include ethyl acetate,acetone, hexafluorbenzene, methyl ethyl ketone and similar solvents andmixtures and combinations thereof, most preferred is ethyl acetate aloneor in combination with isoamyl acetate. Typical preferred concentrationsinclude about 0.01 to about 0.3 weight percentpoly[bis(trifluoroethoxy)phosphazene or its derivative in solution, morepreferably about 0.02 to 0.2 weight percentpoly[bis(trifluoroethoxy)phosphazene, and most preferably about 0.075 toabout 0.2 weight percent. It should be understood based on thisdisclosure that the type of hydrogel core can be varied as can thetechnique for coating a particle, however it is preferred that a corewhich is useful in the treatment techniques and applications describedherein is formed and subsequently coated with poly[bis(trifluoroethoxy)phosphazene and/or its derivatives as described herein.

As previously discussed, the particles can be used in various medicaland therapeutic applications, such as embolization, drug delivery,imaging (ultrasound) and as tracer particles. For example, in oneembodiment, the invention includes a method of minimizing blood flow toa specific tissue in a mammal. This process, commonly referred to asembolization, includes occluding or obstructing at least a portion of avessel, or the entire vessel, with one or more of the particles of theinvention. Such procedure is particularly useful in the treatment ofdiseases and pathologies that involve undesirable vascularized tissues,for example, tumor tissue or disorders involving the uncontrolledproliferation of certain cells such as endometriosis. In suchprocedures, the particle(s) are prepared in accordance with theprocedures described above, and may be inserted into the blood vessel byany invasive or non-invasive medical practice known or to be developedin the art such as via a catheter, a syringe, or a surgical incision.The embolization can be carried out such that only a portion of theblood vessel is occluded, or the entire vessel may be occluded. In themethod, if desired, one may use particles that have been loaded with anactive agent, such as a cytostatic agent, an anti-inflammatory agent, ananti-mitogenic or cell proliferation active agent, a hormone, or anyother desirable active agent, as described herein. Embolizationparticles according to the present invention are capable ofdemonstrating improved optical visibility, additional radiopacity, andan optimum specific density of about 1.17 g/cm³. The embolizationparticles in this invention may be used with different dyes as markersas noted above for particle sizes, embedded pharmaceuticals forlocalized drug delivery and controlled drug elution characteristics.

For use in embolization therapy, particle density is preferably takeninto consideration to ensure beneficial properties for particledelivery. Possible clogging of a catheter-based delivery system mayoccur if using a density-mismatched delivery medium. In addition, it isdesirable to include a certain minimum amount of contrast agent in thedelivery medium to achieve sufficient levels of fluoroscopic contrastduring surgery. Currently, the polymethacrylate hydrogel density isbetween 1.05 g/cm³ and 1.10 g/cm³ depending on the equilibrium watercontent. The most common iodinated nonionic contrast agent media with300 mg iodine per ml have densities of 1.32-1.34 g/cm³. As used herein,“buoyancy” refers to the ability of the particles to be substantiallyfree floating in solution that occurs when the density of the particleis substantially the same as the medium in which it is suspended. Coatedparticles formed in accordance with the present invention as describedherein can reach buoyancy when there is approximately 30% contrast agentin the delivery medium, however, such levels can be adjusted for suchpreferred use according to techniques described herein.

One method for increasing the density of the particles is by use ofheavy water or deuterium oxide (D₂O). When heavy water is used to swellthe particles, D₂O displaces H₂O, thereby increasing the weight of theparticles for better dispersion and buoyancy levels. Typically thisleads to the ability to add higher amounts of contrast agent of at leastabout 5% using such a technique. However, some equilibrating effect canoccur over time when the particles are contacted with an aqueoussolution of contrasting agent. Thus, it is preferred that when using D₂Ofor this purpose, either that suspension times are kept to a minimum or,more preferably, that the contrast agent be provided in a solution whichalso uses D₂O.

Alternatively, particles of pH 1 can be neutralized with cesiumhydroxide and/or the final neutralized particles can be equilibratedwith cesium chloride. Such compounds diffuse cesium into the particles,such that either the cesium salt of polymethacrylic acid is formed orpolymethacrylic acid is diffused and thereby enriched with cesiumchloride.

The cesium increases the density of the particles, thereby increasingthe ability to add higher amounts of contrast agent. Typical buoyancylevels can be adjusted using the cesium technique such that about 45 toabout 50% contrast agent may be added to the delivery medium as isdesired for embolization. Cesium salts are non-toxic and render theparticles visible using fluoroscopy. Cesium's atomic weight of 132.9g/mol is slightly higher than that of iodine providing beneficialeffects including increase in overall density and enhancement of X-raycontrast visibility even without a contrast agent. For certain cancertreatments where a radioactive isotope of cesium is desired, such activeagent can be used as an alternative cesium source rendering theparticles buoyant in an embolic solution as well as able to be used asan active treatment source.

The above-noted techniques for improving density of particles, such asmicroparticles for embolization or other applications where densityand/or buoyancy in solution are applicable properties may be applied into the preferred particles described herein and/or may be applied forother similar particles. It should be understood that the disclosure isnot limited to cesium and/or D₂O treatment of the preferred particlesherein and that such techniques may have broader implications in otherparticles such as other acrylic-based hydrogels and other polymericparticles.

As noted above, barium sulfate may be used between the core particlesand the preferred poly[bis(trifluoroethoxy) phosphazene coating orintroduced into the interior of the core particles using any techniqueknown or to be developed in the art. Also, organic dyes may similarly beincluded in the particle core. These materials, particularly the bariumsulfate, also contribute to an increase in density as well as providingradiopacity. In addition to a general density increase as provided bythe above-noted D₂O or cesium compounds, the barium sulfate allows thisbenefit even upon substantial and/or full hydration, allowing particlesin suspension to remain isotonic. Thus, a barium sulfate powder coatingcan provide an inert precipitate having no effect on physiologicalosmolarity.

In yet other exemplary embodiments of the present invention, contrastagents for conventional radiographic imaging with radio-opaque agentssuch as cesium, iodine, or ionic or nonionic iodine-containing compoundsmay be provided either within the hydrogel core or injected as asuspension liquid with the microspheres. Examples of iodine contrastagents include both ionic and non-ionic agents, including, but notlimited to, Diatrizoate, Metrizoate, Ioxaglate, Iopamidol, Iohexol,Ioxilan, Iopromide, Iodixanol, Ioxitalamin, and the like. Generally, theterm “radio-opaque” agent refers to any substance or agent which blocks,absorbs, scatters, or reflects any radiation outside the visible lightspectrum, including, but not limited to, X-rays (in the wavelength rangeof 0.01 to 10 nm), beta rays (having, for example, velocities of about35,000 to 180,000 miles per second), gamma rays (having an energy in therange of 10⁴ to 10⁷ eV), radiation used in radiation therapy (forexample, therapy to treat cancer), and other harmful radiation (such asthat resulting from nuclear disasters and nuclear weapons). Suitableradio-opaque agents include, but are not limited to, those comprisingplatinum, gold, silver, bismuth, mercury, lead, barium, calcium, zinc,aluminum, iron, gallium, iodine, tungsten, and any combination thereof.Other suitable radio-opaque agents include, but are not limited to,those commercially available as radio-opaque agents for medical uses,such as ionic and nonionic intravenous radiocontrast agents, diagnosticbarium and gastrographin preparations, and gallium preparations. In oneembodiment, the radio-opaque agent blocks, absorbs, scatters, orreflects any radiation outside the visible light spectrum, including,but not limited to, X-rays, beta rays, and gamma rays, which are emittedfrom radioisotopes, such as those used in the medical industry (forexample, in radiation therapy and medical diagnostic testing). Examplesof radioisotopes in clinical use include, but are not limited to,radioisotopes of gallium (for example, ⁶⁷Ga or ⁶⁸Ga), iodine (forexample, ¹²³I, ¹²⁶I, ¹³¹I, ¹³²I, or ¹³³I), indium (for example, ¹¹¹In or¹¹³In), thallium (for example, ²⁰¹Tl or ²⁰³Tl), as well as ³H, ¹¹C, ¹⁴C,¹³N, ¹⁸F, ²²Na, ²⁴Na, ³¹Si, ³²P, ³⁵S, ³⁶Cl, ³⁸Cl, ⁴²K, ⁴⁵Ca, ⁵¹Cr, ⁵²Mn,⁵⁴Mn, ⁵⁵Fe, ⁵⁹Fe, ⁶⁰Co, ⁶³Zn, ⁶⁵Zn, ⁶⁸Zn, ⁸²Br, ⁸⁵K, ⁸⁵Kr, ⁸⁹Sr, ⁹⁹Tc,^(99m)Tc, ^(99m)Re, ¹⁰¹Re, ¹⁰⁵Re, ^(121m)Te, ^(122m)Te, ^(125m)Re,¹³⁷Cs, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ^(81m)Kr, ³³Xe, ⁹⁰T, ²¹³Bi, ⁷⁷Br, ¹⁸F, ⁹⁵Ru,⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ¹⁸²Ta, ¹⁹²Ir, ¹⁹⁸Au, and the like.

It should be understood, based on this disclosure, that the variousbuoyancy additives noted above can be used independently or incombination to provide the most beneficial effects for a given coreparticle and coating combination.

The invention also includes methods of delivering an active agent to alocalized area within the body of a mammal. The method includescontacting the localized area with at least one of the particles of theinvention as described above, such that an effective amount of theactive agent is released locally to the area. As used herein, “contact”or “contacting” the localized area with at least one particle isintended to include situating at least one particle and an active agentin sufficiently close proximity to the intended area such that thedesired effect is achieved. In this example an effective amount of theactive agent is exposed to the localized area. Diseases or pathologiesthat may be treated by this method include any wherein the localized ortopical application of the active agent achieves some benefit incontrast to the systemic absorption of the drug. Suitable active agentsinclude NSAIDS, steroids, hormones, nucleic acids, agents used in thetreatment of disorders of the gastrointestinal tract, such as, ulcers,Crohn's disease, ulcerative colitis, and irritable bowel syndrome. Otheractive agents may include tacrolimus, sirolimus, paclitaxel,cis-/carboplatins, antineoplastic agents, doxorubicine and/or receptorblocking agents, for example, avβ3 integrin blockers, which inhibit cellattachment.

If the particle formulated for delivery of an active agent to alocalized area is about 1 to about 1,000 μm in diameter, the drug loadedmicrospheres can be applied to localized areas within the mammalian bodyusing syringes and/or catheters as a delivery device, without causinginadvertent occlusions. For example, using a contrast agent, a cathetercan be inserted into the groin artery and its movement monitored untilit has reached the area where the localized administration is desired. Adispersion of the particles in a suitable injection medium can beinjected through the catheter, guaranteeing only a specific area of thebody will be subjected to treatment with drug loaded beads (particles).As will be understood to a person of skill in the art, injection mediumsinclude any pharmaceutically acceptable mediums that are known or to bedeveloped in the art, such as, for example, saline, PBS or any othersuitable physiological medium. In accordance with a further embodimentdescribed herein, the invention includes an injectible dispersionincluding particles and a contrasting agent which particles aresubstantially dispersed in the solution. In a preferred embodiment, theparticles are also detectible through fluoroscopy.

The polymeric particles of the invention may be used to prepare asustained release formulation of an active agent for oraladministration. The formulation comprises a particle, as describedabove, loaded with an active agent. The polymeric particle utilized maybe hollow, substantially hollow or solid. The particle can be loadedwith the active agent either by dispersion or solvation of the activeagent in the polymer solution prior to the production of micro-sizedparticles through spray droplets, pastillation of a polymer melt orcarrying out of a cryoextraction process. Alternatively, an unloadedpolymer particle can be prepared and subsequently immersed in solutionscontaining active agents. The particles are then incubated in thesesolutions for a sufficient amount of time for the active agent todiffuse into the matrix of the polymer. After drying the particles, theactive agent will be retained in the polymer particle. If this loadingmechanism is utilized, drug loading can be controlled by adjusting drugconcentrations of the incubation medium and removing the particles fromthe incubation medium when an equilibrium condition has been attained.

Moreover, it is envisioned that the active agent can be selected so asto complement the action of the particles in a synergistic fashion,especially if the particles are being used in an occlusive orembolization procedure. For example, if the tissue to which one wishesto minimize blood flow is a tumor tissue, one may wish to load theparticles used in the occlusion with a cytostatic drug, antiangiogenicagents, or an antimitotic drug.

Also provided is a method of tracing the passage of a particle through ablood vessel or other cavity in a mammalian body. The method includesinjecting into the vessel, cavity, or a conduit adjacent to such cavityor vessel, at least one particle, wherein the particle is at least aparticle prepared in accordance with the procedures described above, andwherein the particle further comprises a component to allow its locationto be detected visually, electronically, or by other imaging means.

In a medical setting, a contrast agent is any substance that is used toenhance the visibility of structures or fluids within the body. Inexemplary embodiments of the present invention, the particles mayinclude a contrast agent that may aid in the visualization of theparticle as it passes through the body cavity, blood vessel, and/orother locale. In general, in this application smaller particles arepreferred, such as those in the range of about 1 to about 10 μm,especially if the particles are to be injected into the bloodstream.However, the particles may be of any size so long as, for this purpose,they are not large enough to occlude the blood vessel, body cavity, oradjacent cavity or vessel to which the procedure is being applied.

In various preferred embodiments of the present invention, contrastagents may be provided within particles of the invention as describedherein. In other preferred embodiments of the present invention,contrast agents may be delivered along with particles of the inventionas a susepension or delivery medium. In yet other preferred embodimentsof the present invention, contrast agents may be delivered in a separateinjection or instillation than the particles of the invention.

If the particles are loaded with a contrast agent, their movement can bevisualized with X-ray machines, or any other imaging modality, dependingon the contrast agent utilized. However, if the particles do not containa contrast agent, the flow of the particles may be visualized using¹⁹F-NMR based computer tomography.

If desired, one may coat particles containing a contrast agent with apolymer coating. The polymer coating may comprise any polymer known orto be developed in the art, including any phosphazene polymers. In apreferred embodiment of the present invention, the polymer coating forparticles is poly[bis(trifluoroethoxy) phosphazene or a derivativethereof. If there is any toxicity or concern of toxicity with respect tothe contrast agent, it is desirable that the one or more coating isnon-biodegradable. Depending on the nature of the visualizationprocedure, such contrast agents may be provided (for example, from theclass of conventional radiographic contrast enhancing agents such asionic or nonionic iodine-containing compounds (Imeron™, Optiray™, etc.).

In conventional or x-ray based radiography, contrast agents may beeither positive or negative, Positive contrast agents have a higherattenuation density than the surrounding tissue. This means that thecontrast agent looks more opaque than the surrounding tissue when seenon an x-ray. Negative contrast media has a lower attenuation densitythan the surrounding tissue. This means that the contrast looks lessopaque than the body. Negative contrast is only found as a gas. Positivecontrast agents are substances with high atomic numbers, that are alsonon-toxic.

Positive contrast agents which may be used in some embodiments of thepresent invention include, but are not limited to, agents comprisingbarium, cesium, iodine, gadolinium, tantalum, and compounds orcompositions containing these elements. Negative contrast agents occuras nontoxic gases, including but not limited to, air, oxygen, carbondioxide, nitrogen, helium, argon, xenon, and mixtures thereof.

Contrast agents for Magnetic Resonance Imaging (MRI) are commonly usedin diagnostic imaging. These agents improve the resolution of MRI imagesby increasing the brightness in various parts of the body where theagent resides. Most contrast agents that have been approved for humanuse are extracellular. Where magnetic resonance imaging (MRI) isemployed for visualization of particles of the present invention, thecontrast agent to be provided may be chosen from the class of rare earthcompounds, such as gadolinium-, tantalum-, and samarium-chelates andother compounds and compositions, as is known to the art. Gadolinium(and other lanthanide)-based agents are heavier and better able toabsorb high-energy X-rays than iodine. Gadolinium-based contrast agentshave a relatively short residence time in the vascular system.

More recently, intracellular agents have been introduced that havelonger residence times and allow extended imaging procedures without theneed for repeated injections of the agent.

Extracellular fluid (ECF) agents include products such as Magnevist,Prohance, and Omniscan. These agents are generally nonionic and a recentreport, points out that the development of nonionic contrast agents forMRI has paralleled that for iodinated contrast materials. Ionic chelatesare also hyperosmolar and some of their side effects may be attributedto this property. Gadodiamide (Omniscan®, Winthrop Pharm.) is a nonioniccomplex with 40% osmolality of Gd-DTPA. It has a median lethal dose of34 mmol/kg resulting in a safety ratio of 2-3 times that of Gd-DOTA, and3-4 times that of Gd-DTPA. No abnormal serum bilirubin levels occur;however, elevated serum iron levels have been reported in some patients.The efficacy of this contrast is similar to that of Gd-DTPA. Gadoteridol(Prohance®, Squibb) is the third intravenous contrast agent on themarket. It is a low osmolar, nonionic contrast, as is Gadodiamide.Indications for use and efficacy are similar to the other agents.

Three types of intravascular contrast agents have been developed:Gd-DTPA labeled albumin, Gd-DTPA labeled dextran, and chromium-labeledred blood cells. Intravascular contrast agents normally remain confinedto the intravascular space, compared to Gd-DTPA which distributesthroughout the extracellular fluid space. This is a result ofintravascular agents having a molecular weight of approximately 70,000and above, compared to a molecular weight of 590 for Gd-DTPA. There areseveral advantages of intravascular agents. They can assess perfusion inareas of ischemia and provide information about capillary permeabilityin areas of reperfusion. They can show the extent of tumorneovascularity and associated permeability changes. Finally, they areuseful in studies requiring prolonged imaging.

Since the hydrogel core component in embodiments of the presentinvention may be derived from an anionic hydrogel polymer, such aspolymethacrylic acid and the like, the incorporation of multivalentmetal compounds, including aforementioned rare earth or other metals,may facilitate a favorable ionic interaction of these compounds, such asby ionic crosslinking or similar ionic interaction, thus providing forfavorable retention or accumulation of these compounds in the particlesand hence providing for a sustained release effect of such compounds invarious embodiments according to the present invention.

Yet another class of contrast agents are Ultrasmall Supermagnetic IronOxide Particles (USPIOs). small particles of ferrite used asparamagnetic contrast medium in MR imaging. These agents exhibit strongT1 relaxation properties, and due to susceptibility differences to theirsurroundings also produce a strongly varying local magnetic field, whichenhances T2 relaxation to darken the contrast media-containingstructures. As particulate matter they are taken up by thereticuloendothelial system. Very small particles of less than 300 μmalso remain intravascular for a prolonged period of time and thus canserve as blood pool agents. The agents are also known by theabbreviation SPIOs (“small particle iron oxides” or “superparamagneticiron oxides”) and USPIOs (“ultrasmall particle iron oxides” or“ultrasmall superparamagnetic iron oxides”). SPIOs have been used asdarkening contrast agents for liver imaging and for darkening the bowel.

In yet other alternate preferred embodiments of the present invention,the particles may comprise radioisotopes or radionuclides as traceragents. Such radioisotopes may be localized and identified visuallyusing conventional or computerized scintillation or beta/gamma cameratechnology. Using image fusion or co-registration techniques,scintillation images can be superimposed on images from modalities suchas CT or MRI to highlight the anatomic region of the body in which theradiopharmaceutical is concentrated. Most diagnostic radionuclides usedin medical imaging emit gamma rays, while the cell-damaging propertiesof beta particles may be used in therapeutic applications. Commonradioisotopes used in embodiments of the present invention include, butare not limited to, technetium-99m, iodine-123 and -131, thallium-201,gallium-67, fluorine-Is, and indium-111. However, any radioisotope inclinical use may be used in embodiments of the present invention,including but are not limited to, any radioisotope of gallium (forexample, ⁶⁷Ga or ⁶⁸Ga), iodine (for example, ¹²³I, ¹²⁶I, ¹³¹I, ¹³²I, or¹³³I), indium (for example, ¹¹¹In or ¹¹³In), thallium (for example,²⁰¹Tl or ²⁰³Tl), as well as ³H, ¹¹C, ¹⁴C, ¹³N, ¹⁸F, ²²Na, ²⁴Na, ³¹Si,³²P, ³⁵S, ³⁶Cl, ³⁸Cl, ⁴²K, ⁴⁵Ca, ⁵¹Cr, ⁵²Mn, ⁵⁴Mn, ⁵⁵Fe, ⁵⁹Fe, ⁶⁰Co,⁶³Zn, ⁶⁵Zn, ⁶⁸Zn, ⁸²Br, ⁸⁵K, ⁸⁵Kr, ⁸⁹Sr, ⁹⁹Tc, ^(99m)Tc, ^(99m)Re,¹⁰¹Re, ¹⁰⁵Re, ^(121m)Te, ^(122m)Te, ^(125m)Re, ¹³⁷Cs ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ^(81m)Kr, ³³Xe, ⁹⁰Y, ²¹³Bi, ⁷⁷Br, ¹⁸F, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ¹⁸²Ta, ¹⁹²Ir, ¹⁹⁸Au, and the like.

The invention also includes the method of carrying out an enhancedultrasound imaging procedure (sonography). In order to do this, oneadministers to the ultrasound subject at least one hollow microcapsuleto the area of the ultrasound subject that one wishes to visualize. Suchadministration can be accomplished by any means known or to be developedin the art, including by use of a syringe, catheter or other invasive ornon-invasive medical device, and/or by a surgical incision. In suchmethod, it is preferable to use particles which are hollow orsubstantially hollow, i.e. having a gas-filled inner cavity that isequal to at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 80%, at least about 90%, of the volumeof the entire particle. The hollow particles are administered to aportion of the ultrasound subject which one wishes to image. Suchparticles enhance the ultrasound image by increasing the echogenicityresulting from use of diagnostic ultrasound modalities due to theirabrupt density change, when compared to the surrounding tissue. Thehollow cavities of the particles act to reflect the ultrasound, therebyenhancing the image.

In still other embodiments of the present invention, inclusion ofgas-filled microbubbles provide improved visualization of microspheresin tissue when used in contrast-enhanced ultrasound (CEUS). CEUS is theapplication of ultrasound contrast agents to traditional medicalsonography. Microbubbles have a high degree of echogenicity, which isthe ability of an object to reflect the ultrasound waves. Theechogenicity difference between the gas in the microbubbles and the softtissue surroundings of the body is immense. Thus, ultrasonic imagingusing microbubble contrast agents enhances the ultrasound backscatter,or reflection of the ultrasound waves, to produce a unique sonogram withincreased contrast due to the high echogenicity difference.Contrast-enhanced ultrasound can be used to image blood perfusion inorgans, measure blood flow rate in the heart and other organs, and hasother applications as well.

Coating material selection for particles containing microbubblesdetermines how easily the particle is taken up by the immune system. Amore hydrophilic material tends to be taken up more easily, whichreduces the microbubble residence time in the circulation. This reducesthe time available for contrast imaging. The microbubble's shellmaterial also affects microbubble mechanical elasticity. The moreelastic the material, the more acoustic energy it can withstand beforebursting

The gas content of microbubbles in particles of the present invention isimportant when employed for ultrasound contrast because the gasselection largely determines echogenicity. When gas bubbles are caughtin an ultrasonic frequency field, they compress, oscillate, and reflecta characteristic echo—this generates the strong and unique sonogram incontrast-enhanced ultrasound. Gas cores can be composed of air, or heavygases like perfluorocarbon, or nitrogen. Heavy gases are lesswater-soluble so they are less likely to leak out from the microbubbleto impair echogenicity Therefore, microbubbles with heavy gas cores arelikely to last longer in circulation

Targeting ligands that bind to receptors characteristic of intravasculardiseases can be conjugated to microbubbles, enabling the microbubblecomplex to accumulate selectively in areas of interest, such as diseasedor abnormal tissues. This form of molecular imaging, known as targetedcontrast-enhanced ultrasound, will only generate a strong ultrasoundsignal if targeted microbubbles bind in the area of interest. Targetedcontrast-enhanced ultrasound can potentially have many applications inboth medical diagnostics and medical therapeutics.

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort can be had to various other aspects, embodiments, modifications,and equivalents thereof which, after reading the description herein, cansuggest themselves to one of ordinary skill in the art without departingfrom the spirit of the present invention or the scope of the appendedclaims.

Further, it is to be understood that this invention is not limited tospecific materials, agents, polyphosphazenes, or other compounds usedand disclosed in the invention described herein, including in thefollowing examples, as each of these can vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects or embodiments and is not intended to belimiting. Should the usage or terminology used in any reference that isincorporated by reference conflict with the usage or terminology used inthis disclosure, the usage and terminology of this disclosure controls.

Unless indicated otherwise, temperature is reported in degreesCentigrade and pressure is at or near atmospheric. An example of thepreparation of a polyphosphazene of this invention is provided with thesynthesis of poly[bis(trifluoroethoxy)phosphazene] (PzF) polymer, whichmay be prepared according to U.S. Patent Application Publication No.2003/0157142, the entirety of which is hereby incorporated by reference.

Also unless indicated otherwise, when a range of any type is disclosedor claimed, for example a range of molecular weights, layer thicknesses,concentrations, temperatures, and the like, it is intended to discloseor claim individually each possible number that such a range couldreasonably encompass, including any sub-ranges encompassed therein. Forexample, when the Applicants disclose or claim a chemical moiety havinga certain number of atoms, for example carbon atoms, Applicants' intentis to disclose or claim individually every possible number that such arange could encompass, consistent with the disclosure herein. Thus, bythe disclosure that an alkyl substituent or group can have from 1 to 20carbon atoms, Applicants intent is to recite that the alkyl group have1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20carbon atoms. In another example, by the disclosure that microsphereshave a diameter of approximately 500 to 600 μm, Applicants includewithin this disclosure the recitation that the microspheres have adiameter of approximately 500 μm, approximately 510 μm, approximately520 μm, approximately 530 μm, approximately 540 μm, approximately 550μm, approximately 560 μm, approximately 570 μm, approximately 580 μm,approximately 590 μm, and/or approximately 600 μm, including any rangeor sub-range encompassed therein. Accordingly, Applicants reserve theright to proviso out or exclude any individual members of such a group,including any sub-ranges or combinations of sub-ranges within the group,that can be claimed according to a range or in any similar manner, iffor any reason Applicants choose to claim less than the full measure ofthe disclosure, for example, to account for a reference that Applicantsare unaware of at the time of the filing of the application.

EXAMPLE 1

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy) phosphazene polymer of a molecular weight3×10⁶ g/mol in the polymer solvent ethyl acetate to obtain a 2% (wily)polymer solution. Four milliliters of this polymer solution was manuallydripped into liquid nitrogen using a 5 ml syringe. This dispersion wasdispensed onto a frozen layer of 150 milliliters of pentane. (See FIG.2.) The cryoextraction was allowed to proceed for three days.Subsequently, polymeric particles were retrieved from the reactionvessel, and were air dried at 21° C.

EXAMPLE 2

Microspheres having a diameter of approximately 350 to 450 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight 3×10⁶g/mol in ethyl acetate to obtain a 1% (wt/v) polymer solution. Fourmilliliters of this polymer solution was manually dripped into liquidnitrogen using a 5 ml syringe. This dispersion was dispensed onto afrozen layer of 150 milliliters of pentane. (See FIG. 2.) Thecryoextraction was allowed to proceed for three days. Subsequently,polymeric particles were retrieved from the reaction vessel and were airdried at 21° C.

EXAMPLE 3

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy) phosphazene polymer of a molecular weight12×10⁶ g/mol in methylisobutylketone to obtain a 2% (wt/v) polymersolution. Four milliliters of this polymer solution was manually drippedinto liquid nitrogen using a 5 ml syringe. This dispersion was dispensedonto a frozen layer of 150 milliliters of a 1:9 (v/v) ethanol/pentanemixture (See FIG. 2.). The cryoextraction was allowed to proceed forthree days. Subsequently, polymeric particles were retrieved from thereaction vessel, and dried under reduced pressure at 21° C.

EXAMPLE 4

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight 9×10⁶g/mol in isoamylketone to obtain a 2% (wt/v) polymer solution. Fourmilliliters of this polymer solution was manually dripped into liquidnitrogen using a 5 ml syringe. This dispersion was dispensed onto afrozen layer of 150 milliliters of pentane. (See FIG. 2.) Thecryoextraction was allowed to proceed for three days. Subsequently,polymeric polymers were retrieved from the reaction vessel and driedunder reduced pressure at 21° C.

EXAMPLE 5

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight16×10⁶ g/mol in cyclohexanone to obtain a 2% (wt/v) polymer solution,Four milliliters of this polymer solution was manually dropped intoliquid nitrogen using a 5 ml syringe. This dispersion was dispensed ontoa frozen layer of 150 milliliters of a 1:1 (v/v) ethanol/diethyl ethermixture. (See FIG. 2.) The cryoextraction was allowed to proceed forthree days. Subsequently, polymeric particles were retrieved from thereaction vessel and dried under reduced pressure at 21° C.

EXAMPLE 6

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight 3×10⁶g/mol in ethyl acetate to obtain a 2% (wt/v) polymer solution. Fourmilliliters of this polymer solution was manually dripped into liquidnitrogen using a 5 ml syringe. This dispersion was dispensed onto afrozen layer of 150 milliliters of hexane. (See FIG. 2.) Thecryoextraction was allowed to proceed for three days. Subsequently,polymeric particles were retrieved from the reaction vessel and airdried at 21° C.

EXAMPLE 7

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight 3×10⁶g/mol in ethyl acetate to obtain a 2% (wt/v) polymer solution. Fourmilliliters of this polymer solution was manually dripped into liquidnitrogen using a 5 ml syringe. This dispersion was dispensed onto afrozen layer of 150 milliliters of ethanol. (See FIG. 2.) Thecryoextraction was allowed to proceed for three days. Subsequently,polymeric particles were retrieved from the reaction vessel and airdried at 21° C. The particles were noticeably gel-like and after dryingwere ellipsoid in shape.

EXAMPLE 8

Microspheres having a diameter of approximately 500 to 600 μm wereprepared. First, a polymer solution was prepared by dissolvingpoly[bis(trifluoroethoxy)phosphazene polymer of a molecular weight 3×10⁶g/mol in ethyl acetate to obtain a 2% (wt/v) polymer solution. Fourmilliliters of this polymer solution was manually dripped into liquidnitrogen using a 5 ml syringe. This dispersion was dispensed onto afrozen layer of 150 milliliters of diethylether. (See FIG. 2.) Thecryoextraction was allowed to proceed for three days. Subsequently,polymeric particles were retrieved from the reaction vessel and airdried at 21° C. The resultant particles were, after drying, compact anduniformly spherical.

EXAMPLE 9

A two liter cryovessel as shown in FIG. 6 was filled with 100milliliters of diethyl ether as a non-solvent. Liquid nitrogen wasslowly added until the non-solvent froze. The vessel was then filledwith additional liquid nitrogen, until the amount of liquid nitrogenrose approximately 5 to 10 cm when measured vertically above thenon-solvent layer. The vessel was closed with an insulated lid, and asyringe needle connected via Teflon tubing to a syringe pump wasinserted through a small opening in the lid.

The syringe pump as shown in FIG. 7, was used to dispense between 5 to15 milliliters of the 5 to 40 mg/ml polymer solution in ethyl acetate,slowly into the cryovessel. The rate of the pump was adjusted toapproximately 10 milliliters dispensing volume per hour. A Teflon®cylinder with one inlet and one to eight outlets is used to distributethe dispensed volumes into several vessels in parallel. (It ispreferable that the ratio of solvent to non-solvent volume stays below10% (v/v). Otherwise the particles may adhere to one another.) After thepolymer solution was completely dispensed into the vessel, another 100milliliters of non-solvent was slowly poured on top of the liquidnitrogen.

In carrying out this process, it is noted that it is preferable that theneedle tips used for dispensing are small, such as the G33 size.Additionally, the dropping distance should be more than 5 cm, so thatthe droplets aided by gravity immediately sink into the liquid nitrogenupon hitting the surface.

The liquid nitrogen in the vessel was slowly allowed to evaporate,taking approximately one day. The non-solvent slowly began to melt, andthe polymer solution droplets, still frozen, sank into the coldnon-solvent. After another day of incubation, the now gelled polymerbeads (particles) were retrieved from the vessel by simple filtration.They were allowed to dry at room temperature for approximately 30minutes and then were ready for use in any of the applications describedherein.

EXAMPLE 10

The microspheres prepared by the process of Example 1 were examined forshape and surface morphology by optical microscope, scanning electronmicroscope (SEM) and atomic force microscopy. The results of theseanalyses are shown in FIGS. 3A and 3B). FIG. 3A shows the microspheresas they appear using an optical microscope at 4× magnification. FIG. 3Bshows a microsphere as it appears using a scanning electron microscopeat 100× magnification.

It can be seen that surface morphology of the unloaded spheres istypical for semi-crystalline polymers above glass transitiontemperature. Amorphous as well crystalline regions are prevalentthroughout the sample surface. The surface is macroporous in nature,with pore sizes ranging from nanometers to few micrometers in diameter.

Particles loaded with bovine insulin were also analyzed using scanningelectron microscopy (100× magnification). The result of these analysescan be seen in FIG. 4A and FIG. 4B).

EXAMPLE 11

Several polymerizations were carried out using varying combinations ofPMMA and three different crosslinking monomers (EDGMA, DEGDMA andTEGDMA), different radical initiators (benzoyl peroxide (BPO) andlauroyl peroxide (LPO), EDTA as a complexing agent and varyingdispersants (Cyanamer 370M, polyacrylic acid (PAA) and varying types ofpolyvinyl alcohol (PVA) to achieve the preferred core particles. In somepolymerizations, sodium phosphate buffer solution (Na₂HPO₄/NaH₂PO₄) wasused. It was observed that some of the reaction procedures wentunsuccessful due to the type of dispersant and concentration chosen.Failure of the dispersant was demonstrated in the form of early onset ofan exothermic reaction, coalescing aqueous and organic phases andpremature onset of the vitrification phase. Only the successful runs areshown in Table 1, which includes the components, concentrations, andreaction conditions for such samples (1-6).

TABLE 1 Sample 1 2 3 4 5 6 Monomer PMMA PMMA PMMA PMMA PMMA PMMA 99.0 g190.0 g 182.0 g 200.2 g 200.2 g 200.2 g Crosslinker EGDMA EGDMA EGDMADEGDMA TEGDMA TEGDMA (1 wt %/ (1 wt %/ (1 wt %/ (0.5 mol %/ (0.5 mol(0.5 mol %/ monomer) monomer) monomer) monomer) %/ monomer monomer) 7.5mMol DDM) Radical LPO LPO LPO LPO LPO LPO Initiator (0.3 wt % (0.3 wt %(0.3 wt % (0.3 wt % (0.3 wt % (0.3 wt % monomer monomer) monomer)monomer) monomer) monomer) Complexing EDTA EDTA EDTA EDTA EDTA EDTAAgent 22 mg 44 mg 44 mg 56 mg 56 mg 56 mg Monomer/ 1:5 1:5 1:5 1:6 1:61:6 Water Ratio Dispersant PVA 4/88 PVA 4/88 PVA 26/88 PVA 26/88 PVA PVA26/88 35% PVA 35% PVA 0.25 wt %/ 0.23 wt %/ 26/88 0.23 wt 0.23 wt %/26/88 26/88 water water %/ water 65% 1 wt 65% 0.5 wt water %/ %/ waterwater Buffer No No No Yes Yes Yes Solution Reaction 1 h 67° C. 1 h 67°C. 1 h 67° C. 1 h 67° C. 1 h 67° C. 1 h 67° C. Temperature/ 2 h 70° C. 2h 70° C. 2 h 70° C. 2 h 70° C. 2 h 70° C. 2 h 70° C. Time 1 h 80° C. 1 h80° C. 1 h 80° C. 1 h 80° C. 1 h 80° C. 1 h 80° C. Outcome 1-50 μm20-200 μm 100-200 μm 1-100 μm 1-100 μm 50-1,000 μm (particle due to dueto due to due to due to due to size) dispersant dispersant dispersantinitial initial initial conc. conc. conc. stirring at stirring atstirring at 400 rpm 400 rpm 130 rpm

EXAMPLE 12

Hydrogel micropartieles formed in accordance with the proceduresdescribed herein were evaluated for buoyancy and suspension propertiesfor use in embolization applications. The microparticles included asample using unmodified polymethacrylic acid potassium salt hydrogelparticles (Sample A); a sample using trifluoroethyl esterifiedpolymethacrylic acid potassium salt hydrogels (Sample B); and a sampleusing the same hydrogel as Sample B, but wherein the particles werecoated with poly[bis(trifluoroethoxy)phosphazene (Sample C). An isotonicphosphate buffered saline solution of pH 7.4 having 0.05 volume % Tween™20 was prepared by dissolving 5 phosphate buffered saline tablets(Fluka®) in 999.5 ml of milliQ ultrapure water. 0.5 ml of Tween 20™surfactant was added to the solution. Solutions having between 20 and 50percent by volume of Imeron300® contrast agent in the isotonic bufferedsaline solution were then prepared for evaluation.

The contrast agent solutions which were prepared were then placed in 4ml vials in aliquots of 2 ml each. To the vials, 50-80 mg of thehydrated hydrogel Samples A-C were added. Each Sample was first hydratedby adding to 100 mg of dry hydrogel microparticles either 900 mg ofisotonic phosphate buffered saline solution or D₂O to obtain 1 mlswollen hydrogel. Buoyancy properties were measured immediately andevery 10 minutes thereafter until buoyancy equilibrium was achievedand/or surpassed.

All of the particles reached equilibrium density in the contrast agentsolution having 30-40% contrasting agent within 5 min. Particles whichwere swollen with D₂O were heavier within the first 10 minutes, but theD₂O did diffuse out of the particles over time within 15-20 min. ofimmersion. If additional water which could displace the D₂O were notadded, microparticles hydrated with D₂O would be able to increase thecontrast agent percentage achievable with adequate buoyancy by as muchas 5%. Particles began to float to the top over time when the contrastagent was added in percentages of 40%-50%.

The equilibrium buoyancy (matching densities) was achieved for Sample Cin 31±1 volume percent of contrast agent in solution. With regard toSamples A and B, swelling behavior and subsequent density are typicallydependent on crosslinking content, pH, ionic strength and valence ofcations used. However, it was assumed herein that the swelling does notinfluence buoyancy due to the sponge-like nature of the polymethacrylicacid hydrogel material. After such material was coated with thepoly[bis(trifluoroethoxy)phosphazene as in Sample C, a time lag ofswelling was observed and buoyancy equilibrium was slower to achieve.

EXAMPLE 13

In order to take account of the time lag and to achieve a more preferreddensity, as well as to enhance the fluoroscopic visibility of theparticles, cesium treatment was then effected for the types ofmicroparticles used in Samples B and C of Example 12.

100 mg of Sample C and of Sample B were hydrated each for 10 min. in a30 weight percent solution of sodium chloride. The supernatant liquidwas decanted after equilibrium and the microparticles were washedthoroughly with deionized water. They were then equilibrated for another10 min., decanted and suspended in 3 ml of surfactant-free isotonicphosphate buffer solution at a pH 7.4. The effect on buoyancy was thenevaluated using contrast agent solutions varying from 20 to 50% byvolume of Imeron® 300. In this Example, 0.1 g of the microparticles ofSamples B and C were used. 3.5 ml of Imeron 300 contrast agent wereprovided to the initial buffer solution which included 4.0 ml isotonicphosphate buffer/Tween™ 20 solution.

The equilibration procedure using cesium chloride yielded particles ofincreased density. Both microparticle samples showed a final buoyancy inthe Imeron® 300 contrast agent solutions at concentrations of 45-50%contrast agent, regardless of the presence or absence of Tween™ 20surfactant. The conditions for saturation appeared to be dependent uponthe initial pH of the particles, the pH used during the procedure andthe corresponding saturation with methacrylic acid groups in theparticle. At pH below 3.6, constant exchange between protons and cationswas observed. As a result, more beneficial results were shown at pHabove about 3.6 and below about 6.6 to temper the amount of cesium.Within the preferred range. buoyancy can be varied. At reasonablyneutral levels, based on test at pH of 7.4, the microparticles did notlose their buoyancy after storage in the contrast agent bufferedsolution over night.

EXAMPLE 1.4

Further compressibility and mechanical property testing were done onmicrospheres in accordance of Samples B and/or C of Example 12. Apressure test stand which was used for further evaluation is shown inFIG. 8. An automated syringe plunger 2 having a motor 4 for providing avariable feed rate of 0 to 250 mm/h and a gear box 6 was furtherequipped with a Lorenz pressure transducer 8 capable of measuring forcesin the 0 to 500 N range. The syringe plunger 2 was in communication witha syringe body 10 as shown. The digital output of the transducer wasrecorded using a personal computer. The syringe body 10 was filled with5 ml of a solution of contrast agent in isotonic phosphatebuffer/surfactant (Tween™ 20) solution in a concentration of about 30-32volume percent contrast agent. Microparticles were provided to thesyringe as well in an amount of 56 mg dry mass. The syringe contentswere then injected through the microcatheter 12 which was attached tothe distal end 14 of the syringe. The microcatheter had a lumen diameterof 533 μm. The force needed to push the microparticles through thecatheter into the Petri dish 16 (shown for receiving microparticlesolution) was measured and recorded as pressure.

In order to make certain calculations, the following information wasapplied as based on typical use of microspheres for embolization.Typically such microspheres have a water content of about 90% such thata vial for embolization would therefore contain 0.2 mg of embolizationparticles in 9.8 ml of injection liquid (2 ml of hydrated microparticlesin 8 ml supernatant liquid). Standard preparation procedures includeadding 8 ml of Imeron® 300 contrast agent to the contents of a singlevial. This would provide an equilibrium concentration of contrast agentof 8 ml/(9.8 ml+8 ml)=44.9 volume percent within an injection solution.The solution is typically drawn up in 1 ml syringes for final delivery.The injection density thus equals:

ρ=V _(Emb) /V _(Tot)=2 ml/18 ml=0.111 Embolization agent per volumefraction.

The Sample C spheres demonstrated approximately the same equilibriumwater content as typical embolization spheres. To achieve the sameinjection density desired for typical surgical procedures, 56 mg ofSample C microspheres were added to 5 ml of a 31 volume percent contrastagent solution in isotonic phosphate buffer and surfactant as notedabove.

The Sample B and C microspheres were evaluated in differentmicrocatheters of equal lumen diameter at a pH of 7.4. Injections inboth the horizontal and vertical direction were made under differentbuoyancy levels and using different swelling levels (based on pH of 6.0in contrast to pH 7.4). The results demonstrated that as long as thediameter of the microspheres was below the internal diameter of themicrocatheter, the microparticles passed through the catheter withoutadditional frictional force in the same manner as the referencesolution. An increase to about 1.0 to 1.4 kg gravitation force wasmeasured when the microparticle diameter reached the same dimension asthe lumen diameter. At roughly 20% compression, forces of about 1.5-2.3kg were needed to overcome frictional forces within the catheter. Forcesgreater than 5 kg were taken as a guideline for moderate to highinjection pressures. When particles are heavier than the injectionmedium, clogging was observed when injecting in the vertical position.When injecting the microparticles in the horizontal position, it wasobserved that serious clogging was alleviated and that larger volumeswere injectible over time.

Injection pressure was further minimized when a lower pH (reducedswelling) was used in combination with horizontal injection such thatthe injection pressures were comparable to the injection media itself.In addition, injection of Sample C microparticles also exhibited a goodinjection pressure pattern at a physiological pH. The catheter entrancedid not clog and each peak in the curve corresponded to either a singlemicroparticle or number of particles passing through the catheter.

The results of the various catheter simulation tests shows that theinvention can be used to form injectible microparticles having a densitywhich substantially matches the density of the injection medium forembolization use. The particles' compressibility can further be suchthat it can be injected without forces over more than about 5 kg on thesyringe plunger. The pH of the injection medium can be taken down toabout 6 or injections can be done horizontally to increase the ease ofpassage of Sample B and C microparticles through the catheter. Oncewithin the blood stream, the particles can expand to their original sizein the pH 7.4 environment.

Additional swelling tests were conducted on the microparticles of SampleC and it was observed that when ion concentrations were low, swellingincreased. In higher concentrated solutions, swelling decreased.Continued dilution of the microparticles of Sample C in a buffersolution led to an increase from 17% to 20% in size of themicroparticles. When mixed into an isotonic phosphate buffer solution,the microparticles initially increase in size between 83.8 and 97%,wherein in deionized water, size increases are from about 116.2 to about136.6%, referring to the dry particles.

In further testing to evaluate the compressibility of the microparticlesof Sample C, the syringe pressure test stand of FIG. 8 was used,however, an optical microscope was used to evaluate the microparticlesas they passed through a progressively narrowed pipette which wasattached to polyethylene tubing connected to the syringe containing aphosphate buffer solution suspension of microparticles of Sample C. Thepipette narrowed to an inner diameter of 490 μm and the pipette wasmounted to a Petri dish such that the narrowest part was submerged inphosphate buffer solution to avoid optical distortion and to collect theliquid ejected from the pipette during measurement. Optical microscopepictures were taken of the microparticles passing through the pipettebefore and during compression. In observing the microparticles, none ofthem underwent a fracture, nor did they form debris or coatingdelamination after passing through the narrow site. Microparticles whichwere chosen to be deliberately too big for the narrow site (for acompression of about 40%) did not break or rupture, but clogged thenarrow site instead. The maximum compressibility under a reasonableamount of force on the microparticles while still allowing themicroparticles to pass through the catheter was about 38.7%. Based onthese evaluations, the microparticles according to Sample C demonstrateproperties that would allow particles which are too large to clog thecatheter rather than break up and cause potential damage to the patient.The test results provided suggested preferred use parameters for SampleC microparticles for embolization use as shown in Table 2 below:

TABLE 2 Particle Radius s Constriction (μm) (μm) Compression (%) ForceNeeded (kg) 340 540 25.9 and 26.5 2.58 and 1.92 360 540 33.3 3.19 330540 22.2 2.83 330 540 22.2 2.14 370 540 37.0 and 37.3 3.59 and 2.77 330540 22.2 2.08 320 540 18.5 and 18.4 1.61 and 1.38 330 540 22.2 1.71

Sample C microparticles were further subjected to mechanical and thermalstress stability testing. Microparticles, after passing through a TerumoProgreat Tracker catheter were washed with deionized water to removeresidual buffer solution along with contrast agent. They were dehydratedfor 12 h at 60° C. and then transferred to an SEM for surface analysis.They were compared with particles from the original batch ofmicroparticles which had undergone the same hydration/dehydration cyclein milliQ ultrapure water, but which had not been passed through thecatheter. FIGS. 9A and 9B show the surface of the Sample Cmicroparticles just after the hydration/dehydration cycle and the filmthickness of an exemplary Sample C microparticle, respectively. SEMsafter passing through a catheter at various magnifications (FIGS. 10A,10B, 10C and 10D) show that the coating did not delaminate (FIG. 10A).Some microparticles did demonstrate some stretching out in the coatingfilm (FIGS. 10B and 10C). However, a closer magnification as in FIG. 10Ddemonstrates that the morphology of the coating layer is still intact.

A sterilizer was filled with 2 l of deionized water and 10 vials eachhaving 56 mg of Sample C microparticles in 3.3 g of solution of isotonicphosphate buffer/surfactant (Tween™ 20) and turned on. The water boilingpoint was reached about 15 min. after the start of the sterilizer, andtemperature was held at that point for 3 min. to remove air by watervapor. The vessel was then sealed shut to raise pressure and temperatureto 125° C. and 1.2 bar pressure. This took approximately 10 min. Thetemperature was then maintained for 15 min, and then the vessel was shutdown for a cooling phase. A temperature of 60° C. was reached about 30min later, after which the vessel was vented, the samples withdrawn andthe vessel shut tightly. A sample vial was opened, and the supernatantliquid decanted. The microparticles were washed with deionized water.After dehydration, they were subjected to measurement using an SEM. Theresults demonstrated only a small number of delaminated coatings on themicroparticles under such thermal stress (see FIG. 11A in the strongwhite contrast portion). The overall percentage of such microparticleswas only about 5 to 10%. Close up, the film delamination which did occurappears to have occurred along crystalline-amorphous domain boundariesin the poly[bis(trifluoroethoxy)phosphazene coating (see FIG. 11B). Mostof the microparticles showed only minor defects (such as a minorcircular patch being missing), but no damage to the hull of themicroparticles (see FIGS. 11C and 11D).

EXAMPLE 15

Microparticles were formed in accordance with a preferred embodimentherein. A deionized water solution of polyvinyl alcohol (PVA) wasprepared using about 23 g of PVA of weight average molecular weight ofabout 85,000-124,000, which PVA was about 87-89% hydrolyzed and 1000 gwater. A phosphate buffer solution was prepared using 900 g deionizedwater, 4.53 g disodium hydrogen phosphate, 0.26 g sodium dihydrogenphosphate and 0.056 g ethylenediamine tetraacetic acid (EDTA). Methylmethacrylate (MMA) monomer was vacuum distilled prior to use.

Polymerization was carried out in a three-necked, round-bottomed,2000-ml flask with a KPG mechanical stirring apparatus attached. Theflask was also equipped with a thermometer, reflux condenser and apressure release valve with a nitrogen inlet. The polymerization processfurther utilized 100 ml of the PVA solution prepared above, 900 ml ofthe phosphate buffer solution, 0.65 g of dilauroyl peroxide, 200.2 gmethacrylic acid methyl ester and 2.86 g triethylene glycoldimethacrylate.

The PVA and buffer solutions were provided to the reactor flask. Thedistilled MMA and triethylene glycol dimethacrylate were introduced,dilauroyl peroxide then added to the same flask and the components wereagitated to ensure dissolved solids. The reaction flask was flushed withargon and the stirrer speed set to at 150 rpm to produce particle sizesof a majority in the range of 300-355 μm. Stirring continued forapproximate 5 minutes. The stirrer was then set to 100 rpm and argonflushing was discontinued. The reaction flask was then subjected to awater bath which was heated to 70° C. and held at approximately thattemperature for about 2 hours. The temperature of the bath was thenincreased to 73° C. and held for an hour, then the water bathtemperature was raised again to 85° C. and held for another hour. Thestirring and heat were discontinued. The solution was filtered and theresulting polymethylacrylate microparticles were dried in an oven at 70°C. for about 12 hours. The microparticles were subjected to sieving andcollected in size fractions of from 100-150; 150-200; 200-250; 250-300;300-355; 355-400; and 400-450 μm with a maximum yield at 300-355 μm.

The PMMA microparticles thus formed were then hydrolyzed. A portion of100 g 250-300 μm sized microparticles, 150 g potassium hydroxide and1400 g of ethylene glycol were added to a 2000 ml flask, refluxcondenser with drying tube connected, and the mixture was heated at 165°C. for 8 hours for full hydrolysis. The mixture was allowed to cool toroom temperature, solution decanted and the microparticles were washedwith deionized water. The procedure was repeated for other calibratedsizes of microparticles (the following reaction times applied: 300-355micron particles: 10 hours; 355-400 micron particles: 12 hours and400-455 micron particles: 14 hours).

The microparticles were finally acidified with hydrochloric acid to a pHof 7.4, and dried in an oven at approximately 70° C.

EXAMPLE 16

Microparticles formed in accordance with Example 15 were then esterifiedin this Example. For esterification surface treatment, 800 g of driedmicroparticles from Example 15 were weighed in a 2L reaction vessel witha reflux condenser. 250 g thionyl chloride in 1.5 L diethyl ether wereadded under stirring. Stirring was continued at room temperature for 20hours. The solvent and volatile reactants were removed by filtration andsubsequent vacuum drying. Then 500 g trifluoroethanol in 1.5 L etherwere introduced and the suspension stirred for another 20 hours at roomtemperature. The particles were finally dried under vacuum.

EXAMPLE 17

In an alternative surface treatment to Example 16, 800 g driedmicroparticles from Example 15 were reacted with 1140 g trifluoroethanoland 44 g sulfuric acid added as a catalyst. The mixture was stirred for20 hours at room temperature, filtered and dried under vacuum.

EXAMPLE 18

800 g of dry PMMA potassium salt microparticles which were partiallyesterified with trifluoroethanol as described above in Examples 15-16were spray coated with poly[bis(trifluoroethoxy)phosphazene in an MP-1Precision Coater™ fluidized bed coating apparatus (available fromAeromatic-Fielder AG, Bubendor, Switzerland). The particles were pickedup by an air stream (40-60 m³/h, 55° C. incoming temperature) and spraycoated with poly[bis(trifluoroethoxy)phosphazene solution microdropletsfrom an air-fluid coaxial nozzle. The solution composition was 0.835 gpoly[bis(trifluoroethoxy)phosphazene, 550 g ethyl acetate and 450 gisopentyl acetate. It was fed through the nozzle's 1.3 mm wide innerbore at a rate of 10-30 g/min. At the nozzle head, it was atomized withpressurized air (2.5 bar). The total amount of spray solution (3 kg) wascalculated to coat the particle with a 150 nm thickpoly[bis(trifluoroethoxy)phosphazene film.

EXAMPLE 19

The dry potassium salt microparticles of Examples 15-16, which werepartially esterified with trifluoroethanol as described above, werespray-coated with diluted poly[bis(trifluoroethoxy) phosphazene solutionin ethyl acetate in a commercially available fluidized bed coatingdevice (see Example 16). 100 mg of such coated, dried microparticles aswell as 100 mg of uncoated, dried PMA potassium salt microparticleswhich were partially esterified with trifluoroethanol, were immersed inabout 30% aqueous cesium chloride solution, prepared by dissolving 30.0g cesium chloride in 100 ml deionized water. The supernatant liquid wasdecanted after 10 min. equilibrium time and the microparticles werewashed thoroughly with deionized water, equilibrated for another 10min., decanted and suspended in 3 ml surfactant free phosphate buffersolution at a pH of 7.4. Density of the particles in solution wasmeasured for matching density in a contrast agent solution. To each typeof microparticle was added a contrast agent solution which included aratio of 3.5 ml of Imeron® 300 contrast agent (density 1.335 g/ml) and 4ml phosphate buffered saline (density 1.009 g/ml). Both hydrogel typesreached buoyancy at levels of 45-50% contrast agent in solution. Thiscorresponds to an increased density of the microparticles of 1.16 g/ml.

EXAMPLE 20

Microparticles were formed in accordance with the procedure of Example15 with the exception that an exterior barium sulfate coating wasprepared on the microparticles after neutralization of the particles andthe microparticles were not dried after neutralization prior to thebarium sulfate coating step. To prepare the barium sulfate coating, 2500ml hydrated particles were subjected to 2000 ml of 0.5 M sodium sulfate(Na₂SO₄) solution and saturated for 4-12 hours. To the particlesuspension was then slowly added 1950 ml of 0.5 M barium chloride(BaCl₂) solution under stirring at room temperature. After washing withexcess deionized water, the resulting particles in a swollen stateincluded a barium sulfate powder coated surface. The particles were thendried and esterified in the mariner noted above in Example 16. Theparticles were then coated using the fluidized bed process of Example 21below. The resulting microparticles were externally coated with anon-adhesive barium sulfate powder. Barium sulfate coatings prepared inaccordance with this invention and procedure are capable of preventingparticle agglomeration during drying and also increase density. Theconcentration and ratios of barium sulfate may be varied to providedifferent results and a use of an excess of sodium sulfate can minimizeresidual barium chloride. The particles formed in accordance with thisexample were effectively washed with hot water to minimize excess bariumsulfate powder that may contaminate vials, etc. The barium sulfate workseffectively to prevent adhesion of particles prior to drying to assistin fluidization of the hydrated microparticles.

EXAMPLE 21

Fluidized bed coating of barium sulfate powdered beads was performedusing polymethacrylate beads with a surface layer of barium sulfateformed in accordance with Example 20 but an excess of barium chloridewas used such that barium ions diffused inside the core and formed aprecipitate inside the hydrogel core.

In preparing the particles, the same procedure for barium sulfate coatedparticles set forth in Example 20 was repeated with the exception thatthe order of addition was reversed. Thus, 2500 ml hydratedmicroparticles were suspended in 2500 ml deionized water and slowly, 5mol % (200 ml) of a 0.5 M (BaCl₂) were added slowly under stirring. Theaddition was performed within a time period of three minutes to preventirreversible barium acrylate formation taking place. The suspension wasthen immediately quenched with the double amount (400 ml) of 0.5 Msodium sulfate (Na₂SO₄) solution under stirring at room temperature.Afterwards, the particles were washed three times with 2 L of deionizedwater each. This procedure precipitated barium sulfate inside theparticles.

The resulting precipitate was precipitated within the pores of thehydrogel core and could not be removed by multiple washings with water.The particles thus formed were found to have a petinanent increaseddensity in contrast to unmodified particles. The density increase wascontrollable by the molar amount of barium chloride used. Amountsranging from 0-15 mol % of barium chloride were used reproducibly withthis procedure. It was observed during evaluations of this procedurethat, if the time period of addition exceeded 5 minutes, based upon thediffusion speed of barium chloride within the particles, the outer poresof the hydrogel core became irreversibly crosslinked, thereby preventingthe barium sulfate precipitate inside from leaching out. This effect wasvisible by optical microscopy as the “diffusion front” of the bariumsulfate was clearly visible as a white band inside the particle, whereasthe surface remained clear.

Both Examples 20 and 21 provided particles having anti-adhesiveproperties that tend not to agglomerate during drying processes;therefore avoiding surface damage. Generally, such an advantage helpsminimize the amount of particles needed for a fluidized bed procedure asthe particles can be fluidized without being completely dried. Theresidual water content may be increased up to 1:1 based on dry weightwithout agglomeration. The Examples also produce particles withincreased density properties wherein the density change appears to bepermanent.

It should also be understood according to this disclosure that generallywhen applying the procedures noted herein, barium sulfate may beintroduced in accordance with the invention in a range of from 0 toabout 100 mol %, and preferably 0 to about 15 mol % to provide particlesthat have preferred elasticity, density and mechanical stabilityproperties.

The particles formed according to this Example having a barium sulfateload inside the core were then esterified according to Example 16 andvacuum-dried. 300 g of the dry beads were suspended in 300 g water whichwas completely absorbed by the polymethacrylate cores within less than 1min while the barium sulfate powdered particle surface appeared dry andthe particles showed no tendency to agglomerate.

The particles (now 600 g) with 50 weight percent (wt %) water insidewere spray coated with APTMS/poly[bis(trifluoroethoxy)phosphazene in anMP-1 Precision Coater™ fluidized bed coating apparatus according toExample 18 with the exception that an additional aminosilane adhesionpromoter was used. The process equipment used was the same as that ofExample 18, but the coating provided included three different layers. Abottom coating of 3-aminopropyltrimethoxysilane (APTMS) adhesionpromoter was provided upon which was a second coating layer of a mixtureof APTMS and poly[bis(trifluoroethoxy)phosphazene and a third, topcoating layer of poly[bis(trifluoroethoxy)phosphazene. All three spraysolutions were prepared by dissolving the coating material in isopentylacetate and ethyl acetate in a 1:1 weight percentage ratio mixture. Thefirst solution included 35 μl APTMS dissolved in 200 g acetate mixture.The second solution included 25 μl APTMS and 125 mgpoly[bis(trifluoroethoxy)phosphazene in 150 mg of the acetate mixtureand the third included 50 mg poly[bis(trifluoroethoxy)phosphazene in 60g of the acetate mixture. The spray solution quantities andconcentrations refer to the coating of a 300 g batch with 350 μmparticles. The absorbed water evaporated at a rate of 5-10 g/min. Theprocess was stopped after 30 min when the coating thickness reached 100nm and the residual water content was 18.4 wt %.

EXAMPLE 22

The absorption of organic dyes was tested on microparticles formedaccording to Example 15. To 2 ml of phosphate buffered saline solutioncontaining 1 ml of hydrated beads was provided an amount of 5-10 μl ofthe respective dye as a 10 millimolar solution in ethanol. The sampleswere incubated for 30-60 minutes at room temperature under gentleshaking of the vial. Supernatant liquid was discarded and particles werewashed three times with 2 ml of either deionized water, saline or PBSbuffer solution prior to visualization with optical and fluorescencemicroscopy. The dyes tested included triphenylmethane derived dyes suchas Fluoescein diacetate and Rhodamin 6G which were evaluated along withcarboeyanine based dyes such as Dil. The triphenylmethane basedFluorecein and Rhoamine dyes exhibited a specific affinity for thehydrophilic PMMA hydrogel core through ionic interactions. They wereable to easily withstand the rigorous conditions of repeated washing andsteam sterilization without substantial leaching.

The carbocyanine dye DiI on the other hand exhibited a high selectivityfor the hydrophobic poly[bis(trifluoroethoxy) phosphazene shell, withoutpenetrating the hydrophilic PMAA core material. Thus with the subsequentstaining employing the combination of DiI and Fluorescein diacetate bothcore and shell could be simultaneously visualized employing afluorescence optical microscope. As a result, this procedure provides afast, sensitive fluorescence-staining assay for the PMAA particles thatmakes core and shell simultaneously visible under conditions encounteredin actual application. This procedure further enables assessment of themechanical-elastic stress or damage to thepoly[bis(trifluoroethoxy)phosphazene shell. It further shows theaffinity of certain classes of dyes for the various components of theparticle.

Use of these and other dyes may be used to visually identify selectedmicrospheres, which may be provided and dyed for identification toindicate certain sizes of microspheres for use in selected clinical ordiagnostic applications. Color-coding may also be used to identifyselected microspheres on the basis of other properties, such as contentof certain therapeutic or diagnostic agents. Applications according tothe present invention may also improve the imaging visualization byenhancing the particles' buoyancy behavior

FIG. 12A shows exemplary microspheres A, B, and C of the presentinvention, in which the microspheres are each of different diameters,and each has a different color-coding. In an exemplary use of suchmicrospheres of the present invention, color-coded microspheres of likesizes may be separately packaged and supplied for use. Such color-codedmicrospheres may provide a user a visual indication of the specificmicrosphere in a particular clinical or diagnostic use.

In various embodiments according to the present invention, microspheresmay be produced in calibrated sizes ranging from about 1 to about 10,000nanometers in diameter. In one embodiment of the present invention,microspheres of the present invention may be provided in sizes of about40, about 100, about 250, about 400, about 500, about 700, and about 900nanometers in diameter, with a visually distinctive color imparted toeach size of microsphere. Other sizes, size ranges, and calibrated sizedmicrospheres lacking color dye are also included in the presentinvention. Not only may the microspheres or particles be provided indifferent size ranges, but their elasticity may be controlled accordingto the present invention to specifically provide for proximal or distalembolization behavior, due to potentially differing ranges ofcompressibility which may alter the traveling distance of the particlesor microspheres upon their release within a selected blood vessel.Microspheres of the present invention may also be provided in customizedsizes and/or with customized colors as specified by a user for specificclinical diagnostic or therapeutic applications.

EXAMPLE 23

Transarterial chemoembolization or TACE is a clinical procedure in whichthe blood supply to a tumor is disrupted by embolization andchemotherapy is administered directly into the tumor. Selectiveembolization of tumor blood vessels without direct administration ofchemotherapy (bland embolization) is also preformed as a clinicalprocedure in certain situations.

In most living organisms with a developed circulatory system, thevasculature tends to taper from larger diameter vessels proximal to theheart to smaller vessels more distal to the heart. Larger arteries thustend to divide into smaller arteries, which eventually taper to thearteriole level and interface with small diameter venules. Venous flowprogresses from such venules through successively larger diameter veinsas flow returns to the heart.

It is common, therefore, that blood vessels of differing sizes may existwithin a tumor mass or other target tissue. In a clinical situationwhere embolization and maximal disruption of blood supply to a tumor orother target tissue is desired, serial embolization of progressivelylarger tumor vessels may provide a more complete embolization, with orwithout the delivery of chemotherapeutic or other therapeutic agents.

FIG. 12B is a conceptual representation of a selective embolization ofan exemplary artery 120 by serial administration of different sizedmicrospheres 121, 122, and 123. The direction of blood flow within theexemplary artery 120 is shown by the arrows in FIG. 12B. In thisexample, microsphere 121 is the smallest diameter of the microspheresadministered, and is injected into artery 120 first, occluding thevessel lumen at the smallest vessel diameter that will not permitpassage of microsphere 121. Continuing in this example, microsphere 122is of intermediate diameter of the microspheres administered, and isinjected into artery 120 first, occluding the vessel lumen at thesmallest vessel diameter that will not permit passage of microsphere122. Finally, in this example, microsphere 123 is the largest diameterof the microspheres administered, and is injected into artery 120 first,occluding the vessel lumen at the smallest vessel diameter that will notpermit passage of microsphere 123. The result in this example is thesequential blockage of blood flow at multiple levels throughout theblood supply of the tumor or target tissue.

In other examples of the present invention, less than three or more thanthree different sized microspheres may be administered to secure thedesired embolization of a tumor or other target tissue.

As provided in previous examples of the present invention,different-sized microspheres of the present invention may further beprovided with color-coding to allow user identification and visualconfirmation of the sized microspheres in use at any given stage of theclinical procedure.

The delivery of microspheres of different sizes or other inherentqualities may further be facilitated by the use of transport packagingand/or delivery devices which are color-coded to allow useridentification and visual confirmation of the sized microspheres in useat any given stage of the clinical procedure in exemplary applicationsaccording to the present invention. In various exemplary applications ofthe present invention, such color-coded devices may be used incombination with color-coding of the microspheres themselves, withcorresponding microsphere and packaging/delivery device color-coding.

FIG. 12C shows a syringe used for the packaging and/or delivery ofcolor-coded microspheres of a select size according to the presentinvention. In the example shown in FIG. 12C, the syringe 124 comprises abarrel 125, a plunger 126, a plunger tip 127, a Luhr-type injection tip128, and a Luhr tip cover 129.

As shown in FIG. 12C, one or more of components barrel 125, a plunger126, a plunger tip 127, a Luhr-type injection tip 128, and a Luhr tipcover 129 may be colored in a common color according to a color code toindicate a desired property of the microspheres contained therein. Inone example of the present invention, a syringe may contain color-codedmicrospheres to indicate a certain microsphere size, and the syringeplunger, plunger tip, and Luhr tip cover may be similarly colored tofurther indicate the desired property of the contained microspheres to auser.

FIG. 12D is a conceptual representation of a selective embolization ofan exemplary tumor mass 130 by intravascular administration ofmicrospheres 135. The direction of blood flow within the exemplaryartery 120 is shown by the arrows in FIG. 12. In this example,microspheres 135 are injected into artery 120 through a catheter 140.The result in this example is the sequential blockage of blood flowthroughout the blood supply of the tumor.

FIG. 12E shows an exemplary particle of the present invention comprisinga microsphere 150 with a polyphosphazene coating 155, further comprisinga core 160 with a gas-filled microbubble 165 for enhanced visibility ofsonography. Particles of this type may be used by administering to anultrasound subject at least one particle comprisingpoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof and acore containing one or more gas filled microbubbles to an area of theultrasound subject to enhance echogenicity therein, and imaging the areaof the subject using ultrasound.

EXAMPLE 24

In conventional radiology, including fluoroscopy, tomography andcomputerized axial tomography (CT) imaging modalities, it is oftenuseful to employ contrast agents to identify certain sites in amammalian body that would otherwise be difficult to distinguish fromadjacent body tissues. This is particularly applicable in the evaluationof tissue that has been embolized, such as a solid tumor mass.

Selective embolization of a solid tumor mass with microspheres of thepresent invention comprising a coating of poly[bis(trifluoroethoxy)phosphazene surrounding a core containing a contrast agent such asbarium sulfate or iodine will permit the visualization of the embolizedmicrospheres on plain x-ray or CT scanning. Serial examinations may showchanges in tumor size following embolization therapy.

EXAMPLE 25

As an alternative embodiment to having a contrast agent within thestructure of microspheres of the present invention, selectiveembolization of a solid tumor mass with microspheres of the presentinvention comprising a coating of poly[bis(trifluoroethoxy)phosphazenemay be achieved using a contrast agent in solution, such as aniodine-containing solution, as a suspension/delivery solution forinjection delivery of the microspheres through a catheter, needle, orcannula placed into arterial blood vessels feeding the targeted tumormass. The presence of the contrast agent will allow visualization of thetumor mass immediately before and after its embolization.

EXAMPLE 26

Selective embolization of a solid tumor mass with microspheres of thepresent invention comprising a coating ofpoly[bis(trifluoroethoxy)phosphazene surrounding a core containing aradioisotope as a tracing agent will permit the visualization of theembolized microspheres on scintillation studies. Depending upon thehalf-life of the radioisotope selected, residual radiation may bedetected in the site for some time, allowing serial examinations whichmay show changes in tumor size following embolization therapy.

EXAMPLE 27

Magnetic Resonance Imaging (MRI) may be used to assess and follow tumorsize following selective embolization of a solid tumor mass withexemplary microspheres of the present invention comprising a coating ofpoly[bis(trifluoroethoxy)phosphazene surrounding a core containing acontrast agent such as tantalum, gadolinium and samarium-chelates, orother rare earth compounds with paramagnetic properties. The response ofsuch a contrast agent to the strong electrical field of the MRI processprovides enhanced imaging results in the targeted, embolized tumortissue.

EXAMPLE 29

At the onset of an interventional embolization procedure, a patientcontaining a targeted tissue is injected by an operator with exemplaryfirst microspheres of the present invention comprising a coating ofpoly[bis(trifluoroethoxy)phosphazene surrounding a core containing acontrast agent. The exemplary microspheres are selected of a relativelysmaller size range, suited for the given medical indication for theprocedure.

The smaller-sized first microspheres in this example may be marked withone or more specific contrast agents covalently bound to the hydrogelcore. Such contrast agents are selected depending upon the imagingtechnology in use. For magnetic resonance imaging (MRI), ionic ornon-ionic contrast agents including, but not limited to tantalum,gadolinium and samarium-chelates, or other rare earth compounds withparamagnetic properties, or MRI active ions, such as Mn or Fe ions.

Once selectively injected under real-time MRI or other imagingvisualization, these specifically marked first microspheres will travelfar more distally in the injected vascular system due to their smallersize.

The interventional procedure is then continued with the administrationof second, larger microspheres of the present invention under real timeimaging control, until the operator identifies the retardation of bloodflow and beginning of vascular saturation in the targeted tissue. Theselarger second microspheres travel more proximally (relative to theadministrator of the microspheres, not the targeted tissue) in theinjected vascular system than the first microspheres previouslyinjected.

To conclude the embolization procedure, the operator may injectdifferently marked third microspheres of the present invention of largersizes. These specifically marked third microspheres will travel moreproximally in the injected vascular system than the first or secondmicrospheres previously injected.

The contrast agent(s) within the first, second, or third microspheres ofthis example may be of different or the same chemical nature, therebymaking it possible to distinguish the first, second, and thirdmicrospheres in situ in real time using MRI or other imaging modalitiesduring the interventional procedure.

The operator may thereby determine the start and endpoints of theembolization procedure, as well as the specific anatomic location of thevascular region embolized, and may further determine the distancebetween most proximal and distal embolization regions and the effect ofdifferent size microspheres in the embolized vascular regions.

The exemplary materials and techniques disclosed herein are applicableto all size ranges of microspheres of the present invention and to allclinical embolization fields. The resulting embolization proceduresoffer superior benefit with increased safety and effectiveness tointracranial, intrathoracic, intraabdominal, retroperitoneal, and otherembolization procedures. These procedures as described may also providevaluable opportunities for radiological research.

In alternate embodiments an operator may inject mixtures of sizedembolic microspheres of the present invention, with each size distinctlymarked with an appropriate contrast agent, Such mixtures of differingsized microspheres may be allowed to settle at random, aiding theoperator in determining the best size range for a needed embolizationand better revealing the local vascular distribution of differentcontrast agents.

MRI contrast agents in exemplary embodiments of the present inventioninclude products such as Magnevist, Prohance, and Omniscan. These agentsare generally nonionic and a recent report, points out that thedevelopment of nonionic contrast agents for MRI has paralleled that foriodinated contrast materials. Ionic chelates are also hyperosmolar andsome of their side effects may be attributed to this property.Gadodiamide (Omniscan, Winthrop Pharm.) is a nonionic complex withtwo-fifths of the osmolality of Gd-DTPA. It has a median lethal dose of34 mmol/kg resulting in a safety ratio of 2-3 times that of Gd-DOTA, and3-4 times that of Gd-DTPA. No abnormal serum bilirubin levels occur,however elevated serum iron levels occurred with an incidence of 8.2% inone study of 73 patients. The efficacy of this contrast is similar tothat of Gd-DTPA. Gadoteridol (Prohance, Squibb) is the third intravenouscontrast agent on the market. It is a low osmolar. nonionic contrast asis Gadodiamide. Indications for use and efficacy are similar to theother agents.

Alternately, contrast agents such as Ultrasmall Supermagnetic Iron OxideParticles or small particles of ferrite or other metallic compounds orparticles comprising Mn, may be used as paramagnetic contrast agents forMR imaging according to the present invention. These contrast agentsexhibit strong T1 relaxation properties, and due to susceptibilitydifferences to their surroundings also produce a strongly varying localmagnetic field, which enhances T2 relaxation to darken the contrastmedia-containing structures. As particulate matter they are taken up bythe RES. Very small particles of less than 300 nanometers also remainintravascular for a prolonged period of time and thus can serve as bloodpool agents. The agents are also known by the abbreviation SPIOs (“smallparticle iron oxides” or “superparamagnetic iron oxides”) and USPIOs(“ultrasmall particle iron oxides” or “ultrasmall superparamagnetic ironoxides”).

The contrast agents within the hydrogel cores of microspheres of thepresent invention may be chosen depending upon the imaging modalityavailable and appropriate for the clinical application. Such imagingmodalities include, but are not limited to, fluoroscopy, computerizedtomography, tomography, scintillation camera, ultrasound, computerizedultrasound, or positive emission tomography. Contrast agents may beselected that provide best visualization with the intended imagingtechnology, and these agents may them be covalently bonded to thehydrogel cores of the microspheres as described herein.

It will be appreciated by those possessing ordinary skill in the artthat changes could be made to the embodiments described above withoutdeparting from the broad inventive concept thereof. It is understood,therefore, that this invention is not limited to the particularembodiments disclosed, but it is intended to cover modifications withinthe spirit and scope of the present invention as defined by the appendedclaims.

We claim: 1.-25. (canceled)
 26. A method of performing a selectiveembolization, the method comprising: a) selecting a targeted tissuewithin a mammalian patient; and b) delivering to the targeted tissue oneor more first particles, the first particles each comprising a core anda coating; wherein the first particle core comprises a firstacrylic-based hydrogel polymer; and the first particle coating comprisesa poly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof. 27.The method of claim 26, wherein the acrylic-based hydrogel polymercomprises a polymethyl methyacrylate hydrolyzed with potassiumhydroxide.
 28. The method of claim 26, wherein thepoly[bis(trifluoroethoxy)phosphazene] ispoly[bis(2,2,2-trifluoroethoxy)]phosphazene or a derivative ofpoly[bis(2,2,2-trifluoroethoxy)]-phosphazene.
 29. The method of claim26, wherein the first particle core comprises a contrast agent.
 30. Themethod of claim 29, wherein the contrast agent is selected fromDiatrizoate, Metrizoate, Ioxaglate, Iopamidol, Iohexyl, Ioxilan,Iopromide, Iodixanol, Ioxitalamin, or a combination thereof.
 31. Themethod of claim 29, wherein the contrast agent is selected from ⁶⁷Ga,⁶⁸Ga, ¹²³I, ¹²⁶I, ¹³¹I, ¹³²I, ¹³³I, ¹¹¹In, ¹¹³In, ²⁰¹T1, ²⁰³T1, ³H, ¹¹C,¹⁴C, ¹³N, ¹⁸F, ²²Na, ²⁴Na, ³¹Si, ³²P, ³⁵S, ³⁶Cl, ³⁸Cl, ⁴²K, ⁴⁵Ca, ⁵¹Cr,⁵²Mn, ⁵⁴Mn, ⁵⁵Fe, ⁵⁹Fe, ⁶⁰Co, ⁶³Zn, ⁶⁵Zn, ⁶⁸Zn, ⁸²Br, ⁸⁵K, ⁸⁵Kr, ⁸⁹Sr,⁹⁹Tc, ^(99m)Tc, ^(99m)Re, ¹⁰⁵Re, 105Re, ^(121m)Te, ^(122m)Te, ^(125m)Re,¹³⁷Cs, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ^(81m)Kr, ³³Xe, ⁹⁰Y, ³Bi, ⁷⁷Br, ¹⁸F, ⁹⁵Ru,⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ¹⁸²Ta, ¹⁹²Ir, ¹⁹⁸Au, or a combinationthereof.
 32. The method of claim 29, wherein the contrast agent isselected from a tantalum compound, a gadolinium compound, asamarium-compound, a paramagnetic rare earth compound, ferric chloride,ferric ammonium citrate, gadolinium-DTPA, gadolinium-DOTA,gadolinium-EDTA, GdCl₃, Gadodiamide, Gadoteridol, gadopentetatedimeglumine, a Cr(III) compound, a manganese compound, Mn(III)TPPS4(manganese(III) tetra-[4-sulfanatophenyl]porphyrin), Fe(III)TPPS4(iron(III) tetra-[4-sulfanatophenyl]porphyrin), manganese dichloride,iron(III) ethylenebis-(2-hydroxyphenylglycine), 99mTc-iminodiacetate,chromium diethyl HIDA meglumine, gadobenate dimeglumine,manganese(II)-dipyridoxal diphosphate, gadolinium oxide, asuperparamagnetic iron oxide, a small particle iron oxide, an ultrasmallsupermagnetic particle iron oxide, or an ultrasmall particle iron oxide.33. The method of claim 26, further comprising: c) confirming deliveryof the first particles to the targeted tissue using a suitable imagingtechnology.
 34. The method of claim 33, further comprising: d)delivering to the targeted tissue one or more second particles, thesecond particles being larger than the first particles, each secondparticle comprising a core and a coating, wherein the second particlecore comprises a second acrylic-based hydrogel polymer; and the secondparticle coating comprises poly[bis(trifluoroethoxy)phosphazene] and/ora derivative thereof; wherein the second acrylic-based hydrogel polymeris the same as or different from the first acrylic-based hydrogelpolymer, and the second particles travel more proximally in the targetedtissue than the first particles.
 35. The method of claim 34, wherein thesecond particle core further comprises a second contrast agent whereinthe second contrast agent is the same or different from the firstcontrast agent
 36. The method of claim 34, further comprising: e)identifying the retardation of blood flow and the beginning of vascularsaturation in the targeted tissue.
 37. The method of claim 34, furthercomprising: f) delivering to the targeted tissue one or more thirdparticles, the third particles being larger than the first and secondparticles, each third particle comprising a core and a coating, whereinthe third particle core comprises a third acrylic-based hydrogelpolymer; and wherein the third particle coating comprisespoly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof;wherein the third acrylic-based hydrogel polymer is the same as ordifferent from the first and second acrylic-based hydrogel polymer, andthe third particles travel more proximally in the targeted tissue thanthe first or second particles.
 38. The method of claim 37, wherein thethird particle core further comprises a third contrast agent wherein thethird contrast agent is the same or different from the first and/orsecond contrast agent.
 39. The method of claim 37, further comprising:g) identifying an endpoint of a desired devascularization in thetargeted tissue.
 40. The method of claim 37, further comprising: g)determining the distance between proximal and distal embolizationregions in the targeted tissue; and h) assessing the effect of thedifferent particles in the embolization regions in the targeted tissue.41. The method of claim 26, wherein the particles are delivered to thetargeted tissue through blood vessels.
 42. The method of claim 26,wherein delivery of the first particle to the targeted tissue results indevascularization of the targeted tissue.
 43. The method of enhancedultrasound imaging, the method comprising: a) orally administering to atargeted tissue of a mammalian patient at least one hollow microcapsulecomprising poly[bis(trifluoroethoxy)phosphazene] and/or a derivativethereof, an active agent, and a density-increasing agent; b) selecting alocalized area within the mammalian patient to be treated with theactive agent; and c) contacting the localized area with one or moreparticles such that the active agent is exposed to the localized area.44. The method of claim 43, wherein the active agent is selected fromthe group consisting of peptides, proteins, hormones, carbohydrates,polysaccharides, nucleic acids, lipids, vitamins, steroids, organicdrugs, inorganic drugs, a cytostatic agent, an anti-inflammatory agent,an anti-mitogenic, and a cell proliferation active agent.
 45. The methodof claim 43, wherein the density-increasing agent is selected from thegroup consisting of heavy water, deuterium oxide (D₂O).cesium compounds,and barium sulfate.